Methods for improving production of biological products by reducing the level of endogenous protein

ABSTRACT

The present disclosure features methods, cells or cell lines, and compositions for increasing the amount of products, e.g., proteins, produced by a cell or cell line by reducing the level of a non-essential endogenous protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to and benefit from U.S. provisional application U.S. Ser. No. 62/697,480 (filed Jul. 13, 2018), the contents of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The disclosure relates to methods and compositions for modifying host cell expression to increase yield of products, e.g., polypeptides, expressed in cultured cells.

BACKGROUND

Cellular products such as recombinant therapeutic proteins are commonly expressed in cell expression systems, e.g., mammalian cell expression systems. Hundreds of market approved biopharmaceuticals are expressed in mammalian cell lines. However, the high cost associated with their production contributes to increasing global health costs.

Accordingly, there is a need to develop and produce methods for the yield of products, e.g., polypeptides, expressed in cultured cells.

SUMMARY

The present disclosure is based, in part, on the discovery that reducing the level of one or more endogenous proteins, e.g., non-essential, redundant, and/or secreted endogenous proteins, in a cell, e.g., production cell, capable of producing a product, e.g., recombinant polypeptide, will increase the yield of the product.

In one aspect, the invention is directed to a method of manufacturing a product, e.g., recombinant polypeptide, in a cell, e.g., a production cell, the method comprising:

reducing the level of an endogenous protein in the cell, e.g., production cell,

thereby manufacturing the product.

In another aspect, the invention is directed to a method of manufacturing a product, e.g., recombinant polypeptide, in a cell, e.g., a production cell, the method comprising:

providing a cell, e.g., production cell,

reducing the level of an endogenous protein in the cell, e.g., production cell, and

culturing the cell under conditions suitable for producing the product, e.g., recombinant polypeptide,

thereby manufacturing the product.

In another aspect, the invention is directed to a cell, e.g., production cell, capable of producing a product, e.g., recombinant polypeptide, wherein the cell, e.g., production cell, comprises a deletion, substitution, or insertion mutation to a copy of the gene encoding an endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the level of expression of the endogenous protein.

In another aspect, the invention is directed to a cell, e.g., production cell, capable of producing a product, e.g., recombinant polypeptide, wherein the cell comprises an siRNA, e.g., capable of binding to a nucleic acid, e.g., mRNA, encoding an endogenous protein or a regulatory element, e.g., promoter or enhancer, operably linked thereto.

In another aspect, the invention is directed to a product, e.g., recombinant polypeptide, produced by a method or cell described herein.

In another aspect, the invention is directed to a pharmaceutical composition comprising a product, e.g., recombinant polypeptide, described herein.

In another aspect, the invention is directed to a siRNA capable of binding to a nucleic acid, e.g., mRNA, encoding an endogenous protein selected from Table E5.

In embodiments, a product, e.g., polypeptide, is one of the polypeptides provided in Table 1 and 2, or variants thereof.

In embodiments, the product is a polypeptide. In embodiments, the polypeptide is an antibody, e.g., a monoclonal antibody, e.g., an IgG1 antibody, or a polynucleotide encoding the antibody. In embodiments, one or more cysteines in the antibody-encoded polynucleotide is replaced with another amino acid. In some embodiments, the one or more cysteines are replaced with one or more serine residues.

In one aspect, the present disclosure features a method for producing a product described herein in a cell, e.g., a recombinant host cell, e.g., a production cell. In an embodiment, the product is a polypeptide, e.g., a recombinant polypeptide.

Examples of products that can be produced using any of the methods or compositions described herein include recombinant products, or products in which at least one portion or moiety is a result of genetic engineering. Recombinant products described herein are useful for diagnostic or therapeutic purposes. In one embodiment, a product comprises a polypeptide, such as an antibody molecule (e.g., a bispecific or multiformat antibody molecule), a fusion protein, or a protein-conjugate. The methods and compositions described herein may be particularly useful for products that are difficult to produce, e.g., in high quantities or with sufficient quality for commercial or therapeutic use, such as next generation biologics (e.g., fusion proteins, bispecific or multiformat antibody molecules, multimeric proteins, and glycosylated proteins). In one embodiment, a cell as described herein, e.g., for producing the product, expresses the product. In one embodiment, the cell comprises an exogenous nucleic acid that encodes a product described herein, e.g., a polypeptide selected from Tables 1, 2, 3, or 4. Additional examples of products are described in the section titled “Products”.

The methods described herein also can result in improved quality of the product as compared to a cell not subjected to lowered temperature. Improvements in the quality of the product can be characterized by one or more of: dissociation (e.g., a decrease in the dissociation of a polypeptide into a less active form); aggregation (e.g., a decrease in aggregates or aggregation); proper folding or assembly (e.g., a decrease in misfolded or unfolded products; or partially assembled or disassembled products); post-translation modification (e.g., increase or decrease in glycosylation heterogeneity, higher percentage of desired or predetermined post-translational modifications); fragmentation (e.g., a decrease in fragmentation); disulfide bond scrambling (e.g., a decrease in undesired isoforms or structures due to disulfide bond scrambling). In one embodiment, the quality of the product, e.g., recombinant polypeptide, is increased, e.g., by 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, e.g., as compared to the quality of product produced by a cell not subjected to lowered temperature.

In embodiments, the method for producing a product as described herein can be performed with one or more additional steps to enhance stability or activity of a polypeptide. These include, but are not limited to: introducing a modification to the production cell that improves ER processing capacity (ER expansion) or secretion; obtaining the product from the cell, or a descendent of the cell, or from the medium conditioned by the cell, or a descendent of the cell; separating the product from at least one cellular or medium component; and/or analyzing the product, e.g., for activity or for the presence of a structural moiety. In one embodiment, the method further comprises a step for improving ER processing capacity (or ER expansion) by introducing a nucleic acid encoding PD1, BiP, ERO, or XBP1. In one embodiment, the method further comprises an additional step for improving secretory capacity or rate of secretion by modulating SNARE machinery or other machinery involved in the secretory pathway, e.g., by introducing a nucleic acid encoding a SNARE component.

Products

Products suitable for the methods described herein include polypeptides, e.g., recombinant proteins; nucleic acid molecules, e.g., DNA or RNA molecules; multimeric proteins or complexes; lipid-encapsulated particles, e.g., virus-like particles, vesicles, or exosomes; or other molecules. In an embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. For example, the recombinant polypeptide can be a difficult to express protein or a protein having complex and/or non-natural structures, such as a next generation biologic, e.g., a bispecific antibody molecule, a fusion protein, or a glycosylated protein.

In any of the methods described herein, the method for producing a product further comprises introducing to the cell an exogenous nucleic acid encoding the product, e.g., polypeptide, e.g., recombinant polypeptide.

In any of the compositions, preparations, bioreactors, or methods described herein, the product, e.g., recombinant polypeptide, is a therapeutic polypeptide or an antibody molecule, e.g., an antibody or an antibody fragment thereof. In one embodiment, the antibody molecule is a monoclonal antibody. In one embodiment, the antibody molecule is a bispecific antibody molecule, e.g., a BiTE (Bispecific T cell Engager), a DART (Dual Affinity Re-Targeting or Redirected T cell).

In one embodiment, the product, e.g., recombinant polypeptide, is selected from Table 1, Table 2, Table 3, or Table 4.

In embodiments, the product is stably expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product, e.g., recombinant polypeptide, is integrated into the chromosomal genome of the cell. Alternatively, the product is transiently expressed by the cell. In one embodiment, the exogenous nucleic acid encoding the product, e.g., the recombinant polypeptide, is not integrated into the chromosomal genome of the cell.

Host Cells

Provided herein are cells, e.g., recombinant cells, e.g., production cells, for producing the products described herein, methods of engineering such cells, and methods for using the cells.

In any of the compositions, preparations, or methods described herein, the cell is a eukaryotic cell. In one embodiment, the cell is a mammalian cell, a yeast cell, an insect cell, an algae cell, or a plant cell. In one embodiment, the cell is a rodent cell. In one embodiment, the cell is a CHO cell. Examples of CHO cells include, but are not limited to, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, CHOZN, or a CHO-derived cell.

In any of the compositions, preparations, or methods described herein, the cell is selected from the group consisting of HeLa, HEK293, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK, VERO, SP2/0, NS0, YB2/0, EB66, C127, L cell, COS, e.g., COS1 and COST, QC1-3, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, and CHOZN.

In one embodiment, the cell is a eukaryotic cell other than a mammalian cell, e.g., an insect, a plant, a yeast, or an algae cell. In one embodiment, the cell is a prokaryotic cell.

In any of the methods or cells, e.g., production cells, described herein, the cell expresses or comprises a product, e.g., a recombinant product, e.g., a next generation biologic selected from a group consisting of a bispecific antibody, a fusion protein, or a glycosylated protein.

In any of the methods or cells, e.g., production cells described herein, the cell is a CHO cell selected from the group consisting of CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, CHOZN, or a CHO-derived cell.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Headings, sub-headings or numbered or lettered elements, e.g., (a), (b), (i) etc, are presented merely for ease of reading. The use of headings or numbered or lettered elements in this document does not require the steps or elements be performed in alphabetical order or that the steps or elements are necessarily discrete from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a graph of cell-specific productivity of Mab in CHO cell lines where a selected non-essential, redundant, and/or secreted endogenous protein was knocked out relative to a CHO cells transfected with a vector lacking gRNAs.

FIG. 1B shows a graph of gene indel success as measured by TIDE analysis of genomic DNA extracted from FACS-sorted pools.

DETAILED DESCRIPTION

The present disclosure features methods and compositions for obtaining higher yields of a product, e.g., a polypeptide, by reducing the level of an endogenous protein, e.g., non-essential, redundant, and/or secreted endogenous proteins, in a cell, e.g., production cell, capable of producing the product. The methods described herein and cells used in said methods show improved quality as compared to the yield and quality obtained from current production methods and/or cells. The methods and compositions described herein are also usable with engineered cells or cell lines with improved productivity, product quality, robustness, and/or culture viability, as compared to the cell expression systems currently used to produce recombinant products.

The methods are suitable for producing next generation biologics. As these methods continue to gain therapeutic utility in patients, the demand for large quantities of next generation biologic products having a high grade of quality for therapeutic use, as well as efficient means for production and efficient development of production cell lines will escalate. Furthermore, many next generation biologics are difficult to express and produce in conventional cell lines using conventional expression techniques known in the art. The current methods are not sufficient to produce these products in the large quantities and at the high grade of quality required for clinical use. The methods described herein comprising reducing the level of endogenous proteins overcome these obstacles, and may be combined with other methods to increase product yield.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a cell” can mean one cell or more than one cell.

“Endogenous”, as used herein, refers to any material from or naturally produced inside an organism, cell, tissue or system.

“Exogenous”, as used herein, refers to any material introduced to or produced outside of an organism, cell, tissue or system. Accordingly, “exogenous nucleic acid” refers to a nucleic acid that is introduced to or produced outside of an organism, cell, tissue or system. In an embodiment, sequences of the exogenous nucleic acid are not naturally produced, or cannot be naturally found, inside the organism, cell, tissue, or system that the exogenous nucleic acid is introduced into. In embodiments, non-naturally occurring products, or products containing portions that are non-naturally occurring are exogenous materials with respect to the host cells described herein.

“Heterologous”, as used herein, refers to any material from one species, when introduced to an organism, cell, tissue or system from a different species. In embodiments, a heterologous material also encompasses a material that includes portions from one or multiple species or portions that are non-naturally occurring. By way of example, in an embodiment, a nucleic acid encoding a fusion protein wherein a portion of the fusion protein is human, a portion of the fusion protein is bacteria, and a portion of the fusion protein is non-naturally occurring, and the nucleic acid is introduced to a human cell, the nucleic acid is a heterologous nucleic acid.

“Peptide,” “polypeptide,” and “protein”, as used interchangeably herein, refer to a compound comprised of amino acid residues covalently linked by peptide bonds, or by means other than peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. In one embodiment, a protein may comprise of more than one, e.g., two, three, four, five, or more, polypeptides, in which each polypeptide is associated to another by either covalent or non-covalent bonds/interactions. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.

“Recombinant product” refers to a product that can be produced by a cell or a cell-free system. The product can be a molecule, a nucleic acid, a polypeptide (e.g., an SS polypeptide), or any hybrid thereof. A recombinant product is one for at which at least one component of the product or at least one nucleotide of a sequence which controls the production or expression of the product, was formed by genetic engineering. Genetic engineering as used herein to generate a recombinant product or a construct that encodes a recombinant product encompasses recombinant DNA expression techniques known in the art (e.g., as described in Current Protocols in Molecular Biology); site-directed, scanning, or random mutagenesis; CRISPR strategies; and zinc finger nuclease (ZFN) strategies. In one embodiment, the recombinant product is a recombinant polypeptide. In one embodiment, the recombinant product is a naturally occurring product. In one embodiment, the recombinant product is a non-naturally occurring product, e.g., a synthetic product. In one embodiment, a portion of the recombinant product is naturally occurring, while another portion of the recombinant product is non-naturally occurring. In another embodiment, a first portion of the recombinant product is one naturally occurring molecule, while another portion of the recombinant product is another naturally occurring molecule that is different from the first portion.

“Recombinant polypeptide” refers to a polypeptide that can be produced by a cell described herein. A recombinant polypeptide is one for which at least one nucleotide of the sequence encoding the polypeptide, or at least one nucleotide of a sequence which controls the expression of the polypeptide, was formed by genetic engineering or manipulation (of the cell or of a precursor cell). E.g., at least one nucleotide was altered, e.g., it was introduced into the cell or it is the product of a genetically engineered rearrangement. In an embodiment, the sequence of a recombinant polypeptide does not differ from a naturally or non-naturally occurring isoform of the polypeptide or protein. In an embodiment, the amino acid sequence of the recombinant polypeptide differs from the sequence of a naturally occurring or a non-naturally isoform of the polypeptide or protein. In an embodiment, the recombinant polypeptide and the cell are from the same species. In an embodiment, the amino acid sequence of the recombinant polypeptide is the same as or is substantially the same as, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from, a polypeptide encoded by the endogenous genome of the cell. In an embodiment, the recombinant polypeptide and the cell are from the same species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a human cell. In an embodiment, the recombinant polypeptide and the cell are from different species, e.g., the recombinant polypeptide is a human polypeptide and the cell is a non-human, e.g., a rodent, e.g., a CHO, other mammalian cell, an insect cell, a plant, a fungi or a bacterial cell. In an embodiment, the recombinant polypeptide is exogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In an embodiment, the recombinant polypeptide is a human polypeptide or protein which does not differ in amino acid sequence from a naturally or non-naturally occurring isoform of the human polypeptide or protein. In an embodiment, the recombinant polypeptide differs from a naturally or non-naturally occurring isoform of the human polypeptide or protein at no more than 1, 2, 3, 4, 5, 10, 15 or 20 amino acid residues. In an embodiment, the recombinant polypeptide differs from a naturally occurring isoform of the human polypeptide at no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues. In embodiments where a portion of the recombinant polypeptide comprises a sequence derived from a portion of a naturally or non-naturally occurring isoform of a human polypeptide, the portion of the recombinant polypeptide differs from the corresponding portion of the naturally or non-naturally occurring isoform by no more than 1, 2, 3, 4, 5, 10, 15, or 20 amino acid residues, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues.

Oxygenation level and level of oxygenation are used interchangeably, and as used herein refer to a level of dissolved oxygen in a volume of liquid or gas, e.g., in a culture. Oxygenation level can be described in units of dissolved oxygen tension (DOT), e.g., in terms of percentage of air saturation (e.g., the percentage of the level of oxygen in air).

A production culture, as used herein, refers to a mixture of growth media and cells (e.g., at least one cell), e.g., recombinant cells, e.g., recombinant host cells expressing a product, e.g., protein (e.g., recombinant polypeptide), cellular factor, e.g., enzymes and metabolites. In some embodiments, a production culture progresses through one or more culture phases (e.g., sequential and/or overlapping culture phases) comprising cell growth/division characteristics. In some embodiments, treatment of a production culture changes as described herein as the production culture progresses through different phases. Production phase, as used herein, refers to a phase of a production culture starting at inoculation and ending upon reaching a specific criteria (e.g., end of a process or time period, e.g., 10-30 days, e.g., 15-20 days) or upon reaching a specific cell viability criteria (e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, or 10% viability, e.g., less than 50% viability). Production phase may include an exponential growth phase in which cell growth and/or division is exponential. Production phase may include a post-growth phase that follows exponential growth phase, in which cell growth and/or division is in the linear phase of a growth curve, e.g., the rate of cell growth and/or division has decreased relative to exponential growth phase. In some embodiments, completion of production phase produces a production mixture from a production culture. In some embodiments, separation of cells from the production supernatant, e.g., harvest, and further separation or purification steps, e.g., post-harvest, are phases that follow the end of production phase.

A production mixture, as used herein, refers to a mixture of product, e.g., recombinant polypeptide, growth media, and cells (e.g., at least one cell), e.g., recombinant cells, e.g., recombinant host cells capable of expressing a product. A production cell is a recombinant host cell capable of expressing a product, e.g., recombinant polypeptide.

Operably coupled, as used herein, describes a relationship between vessels. In an embodiment, culture media or production culture can be transmitted between operably coupled vessels. In an embodiment, flow of culture between operably coupled vessels occurs in a controlled manner, e g, making use of pumps, filters, sensors, means for maintaining or altering culture conditions, and/or means for monitoring flow of liquid.

Operably linked, as used herein, describes a relationship between a first nucleic acid sequence, e.g., a promoter or enhancer sequence, and a second nucleic acid sequence, e.g., a sequence encoding a protein, wherein the first nucleic acid sequence can affect the second nucleic acid sequence, e.g., by affecting transcription, epigenetic modification, and/or chromosomal topology. In some embodiments, operably linked means two nucleic acid sequences are comprised on the same nucleic acid molecule. In a further embodiment, operably linked may further mean that the two nucleic acid sequences are proximal to one another on the same nucleic acid molecule, e.g., within 1000, 500, 100, 50, or 10 base pairs of each other or directly adjacent to each other. In an embodiment, a promoter or enhancer sequence that is operably linked to a sequence encoding a protein can promote the transcription of the sequence encoding a protein, e.g., in a cell or cell free system capable of performing transcription.

In some embodiments, a method of the disclosure comprises or a bioreactor of the disclosure is capable of maintaining a production culture, cells of the production culture, or supernatant from the production culture, at different temperatures throughout different phases. T_(culture), as used herein, is the temperature at which the cells of a production culture are grown, e.g., in growth medium, from the start of inoculation to the end of production phase to produce a production culture in which a protein is produced. In some embodiments, T_(culture) is 30° C.-38° C. T_(production), as used herein, is the first temperature to which the production culture is cooled after post-exponential growth phase. In some embodiments, T_(production) is less than 35° C., 34° C., 33° C., 32° C., 31° C., or 30° C., e.g., 30° C.-33° C. T_(harvest), as used herein, is the second temperature to which the production culture is cooled prior to harvest (e.g., the separation of cells from supernatant). In some embodiments, T_(harvest) is 29° C., 28° C., 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C., 20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C., 11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., or 4° C. In one embodiment, T_(harvest) is 12° C.-18° C., e.g., 15° C. In one embodiment, T_(harvest) is less than 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C. or 24° C. In one embodiment, T_(harvest) is 15° C.±3° C.

A Cas9 molecule or Cas9 polypeptide, as that term is used herein, refers to a molecule or polypeptide that can interact with a guide RNA (gRNA) molecule and, in concert with the gRNA molecule, home or localizes to a site which comprises a target domain and PAM sequence. Cas9 molecule and Cas9 polypeptide, as those terms are used herein, include naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence. Exemplary Cas9 molecule or Cas9 polypeptide sequences can be found in WO2015/157070, hereby incorporated by reference in its entirety. Cas9 molecules or Cas9 polypeptides include Cas9 molecules that have DNA cleaving and nicking activity.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.

Reducing Levels of Endogenous Proteins

The present disclosure, in part, is directed to methods of manufacturing a product, e.g., a recombinant polypeptide, in a cell, e.g., a production cell, wherein the method comprises reducing the level of one or more endogenous proteins in the cell, e.g., production cell. Without wishing to be bound by theory, it is thought that one or more production cell properties (e.g., specific product production rate (qP), growth rate, product yield, and/or viable cell concentration) can be improved by reducing the level of one or more endogenous proteins. Endogenous proteins may consume space and/or resources (e.g., translation, post-translational processing, and secretory space/resources) in the endoplasmic reticulum or Golgi, lowering the capacity available for the production cell to produce product, e.g., recombinant polypeptide. Endogenous proteins may further be redundant with one another in terms of functionality to the cell, e.g., production cell, or be unnecessary to cell viability or growth in the artificial setting of biomanufacturing, e.g., a bioreactor and cell culture.

In some embodiments, reducing the level of one or more endogenous proteins in the cell, e.g., production cell, comprises reducing the level of the one or more endogenous proteins by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% relative to a similar cell that has not had its levels of one or more endogenous proteins reduced. In some embodiments, reducing the level of one or more endogenous proteins in the cell, e.g., production cell, comprises eliminating essentially all, e.g., all, of the one or more endogenous proteins from the cell, e.g., production cell. The level of the one or more endogenous proteins may be assessed, for example, by methods described in Porter, A. J., et al. (2010). “Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: realizing the potential in bioreactors.” Biotechnol Prog 26(5): 1446-1454.

In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves qP. qP, as used herein, refers to specific product production rate. In some embodiments, qP has units of product per unit production cell (e.g., grams or viable cell count) per unit time. In some embodiments, qP is improved, e.g., increased, by at least 1.05× (i.e., 1.05 times), 1.06×, 1.07×, 1.08×, 1.09×, 1.1×, 1.12×, 1.13×, 1.14×, 1.15×, 1.16×, 1.17×, 1.18×, 1.19×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.55×, 1.6×, 1.65×, 1.7×, 1.75×, 1.8×, 1.85×, 1.9×, 1.95×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the qP of a similar cell, e.g., production cell, in which the level of one or more endogenous proteins has not been reduced. In some embodiments, qP is improved, e.g., increased, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to a similar cell, e.g., production cell, in which the level of one or more endogenous proteins has not been reduced. qP may be assessed, for example, by quantification of secreted protein product by ELISA or by quantitative chromatography (e.g., protein A HPLC).

In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves cell growth, e.g., the rate at which cells grow and/or divide. In some embodiments, cell growth is improved, e.g., increased, by at least 1.05× (i.e., 1.05 times), 1.06×, 1.07×, 1.08×, 1.09×, 1.1×, 1.12×, 1.13×, 1.14×, 1.15×, 1.16×, 1.17×, 1.18×, 1.19×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.55×, 1.6×, 1.65×, 1.7×, 1.75×, 1.8×, 1.85×, 1.9×, 1.95×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the cell growth of a similar cell, e.g., production cell, in which the level of one or more endogenous proteins has not been reduced. In some embodiments, cell growth is improved, e.g., increased, by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to a similar cell, e.g., production cell, in which the level of one or more endogenous proteins has not been reduced. Cell growth may be assessed, for example, by temporal measurements of viable cell concentration using automated trypan blue exclusion assays (e.g. using a Beckman coulter Vi-CELL XR).

In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves product yield, e.g., the amount of product a culture produces. In some embodiments, cell growth is improved by at least 1.05× (i.e., 1.05 times), 1.06×, 1.07×, 1.08×, 1.09×, 1.1×, 1.12×, 1.13×, 1.14×, 1.15×, 1.16×, 1.17×, 1.18×, 1.19×, 1.2×, 1.25×, 1.3×, 1.35×, 1.4×, 1.45×, 1.5×, 1.55×, 1.6×, 1.65×, 1.7×, 1.75×, 1.8×, 1.85×, 1.9×, 1.95×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the product yield of a similar culture of similar cells, e.g., production cells, in which the level of one or more endogenous proteins has not been reduced. In some embodiments, product yield is improved by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70% relative to a similar culture of similar cells, e.g., production cell, in which the level of one or more endogenous proteins has not been reduced. Product yield may be assessed, for example, by quantification of secreted protein product by ELISA or by quantitative chromatography (e.g., protein A HPLC).

In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves qP and cell growth. In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves qP and product yield. In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves product yield and cell growth. In some embodiments, reducing the level of one or more endogenous proteins in a cell, e.g., a production cell, improves qP, product yield, and cell growth.

Endogenous Proteins

Endogenous proteins whose levels may be reduced in the methods described herein include those that are non-essential (e.g., to cell growth and/or viability, e.g., in a biomanufacturing setting), redundant functionally with other endogenous proteins whose levels may not be reduced, and/or secreted proteins. A skilled person, based on the disclosure herein and the state of the art, will be able to recognize which endogenous proteins of a cell, e.g., a CHO cell, are non-essential, redundant, and/or secreted proteins with minimal experimentation.

In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a very high level, e.g., as determined by the method of Example 1. In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a high level, e.g., as determined by the method of Example 1. In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a medium level, e.g., as determined by the method of Example 1.

In some embodiments, an endogenous protein whose level may be reduced is a protein associated with functions related with redundancy, non-essentiality, or secretion, e.g., in a protein function database, e.g., the PANTHER database of Example 1. In some embodiments, an endogenous protein whose level may be reduced is associated with a functional keyword listed in Table E1, or a substantially similar functional association. In some embodiments an endogenous protein whose level may be reduced is a protein associated with immune function, disease resistance, cellular communication, and/or secretion.

In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a very high level, e.g., as determined by the method of Example 1, and that is associated with a function related with redundancy, non-essentiality, or secretion, e.g., in a protein function database, e.g., the PANTHER database of Example 1, e.g., is associated with a keyword of Table E1. In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a high level, e.g., as determined by the method of Example 1, and that is associated with a function related with redundancy, non-essentiality, or secretion, e.g., in a protein function database, e.g., the PANTHER database of Example 1, e.g., is associated with a keyword of Table E1. In some embodiments, an endogenous protein whose level may be reduced is a protein that is expressed at least at a medium level, e.g., as determined by the method of Example 1, and that is associated with a function related with redundancy, non-essentiality, or secretion, e.g., in a protein function database, e.g., the PANTHER database of Example 1, e.g., is associated with a keyword of Table E1.

In some embodiments, an endogenous protein whose level may be reduced is not a protein associated with essential functions, e.g., functions that are vital or highly necessary for cell growth, viability, or product production. In some embodiments, an endogenous protein whose level may be reduced is not a protein associated with essential functions, e.g., in a protein function database, e.g., the PANTHER database of Example 1. In some embodiments, an endogenous protein whose level may be reduced is not associated with a functional keyword listed in Table E2, or a substantially similar functional association. In some embodiments an endogenous protein whose level may be reduced is not a protein associated with cellular metabolism, mitochondrial function, cell structure, DNA replication, cell division, checkpoint inhibition/passage, transcription, translation, post-translational modification, protein folding, or the heat shock response.

Exemplary endogenous proteins (and/or the gene encoding said endogenous protein) whose levels may be reduced are listed in Table E5.

In some embodiments, a method disclosed herein comprises reducing the level of more than one endogenous protein. In some embodiments, a method disclosed herein comprises reducing the level of one, two, three, four, five, six, seven, eight, nine, ten, or more (e.g., twenty or more) endogenous proteins (and optionally, up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 endogenous proteins). In some embodiments, a method disclosed herein comprises reducing the level of two or three endogenous proteins, e.g., a combination of endogenous proteins chosen from the combinations of Table E6. In some embodiments, a method disclosed herein comprises reducing the level of two or three endogenous proteins, e.g., a combination of endogenous proteins chosen from those listed in Table E5.

Methods of Reducing Levels of Endogenous Proteins

The present disclosure, in part, is directed to methods of manufacturing a product, e.g., a recombinant polypeptide, in a cell, e.g., a production cell, wherein the method comprises reducing the level of one or more endogenous proteins in the cell, e.g., production cell. In some embodiments, reducing the level of an endogenous protein in the cell, e.g., production cell, comprises eliminating, e.g., knocking out, a copy of the gene encoding the endogenous protein. In some embodiments, reducing the level of an endogenous protein in the cell, e.g., production cell, comprises eliminating, e.g., knocking out, all (e.g., both) copies of the gene encoding the endogenous protein from the genome of the cell. In some embodiments, reducing the level of an endogenous protein in the cell, e.g., production cell, comprises eliminating, e.g., knocking out, a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein. In some embodiments, reducing the level of an endogenous protein in the cell, e.g., production cell, comprises eliminating, e.g., knocking out, all (e.g., both) copies of a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.

In some embodiments, eliminating, e.g., knocking out, comprises introducing a deletion, substitution, or insertion mutation to the gene encoding the endogenous protein or to the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the level of expression of the endogenous protein. In some embodiments, eliminating, e.g., knocking out, comprises introducing a deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8 9, 10, or more base pairs in the gene encoding the endogenous protein or in the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein. In some embodiments, eliminating, e.g., knocking out, comprises substituting 1, 2, 3, 4, 5, 6, 7, 8 9, 10, or more base pairs for different base pairs (e.g., transition or transversion mutations) in the gene encoding the endogenous protein or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.

For example, an insertion mutation that disrupts the reading frame of the gene encoding the endogenous protein or a substitution mutation that alters the start codon of the gene encoding the endogenous protein would both be examples of eliminating, e.g., knocking out, that reduce the level of an endogenous protein. As a further example, eliminating, e.g., knocking out, may comprise insertion of a marker (e.g., an antibiotic resistance, auxotrophy mitigating, or fluorescent marker encoding gene) in a manner that deletes a promoter or portion of a promoter operably linked to the gene encoding the endogenous protein. In some embodiments, eliminating, e.g., knocking out, results in the complete loss of production of the endogenous protein from the copy of the gene encoding the endogenous protein. In other embodiments, eliminating, e.g., knocking out, results in a lower level of production of the endogenous protein, e.g., of functional endogenous protein. In other embodiments, eliminating, e.g., knocking out, results in production of a variant of the endogenous protein that is one or more of: truncated, less-functional, non-functional, misfolded, and/or degradation-prone.

Eliminating, e.g., knocking out, a copy of a gene encoding an endogenous protein or a regulatory element operably linked to said gene may be achieved by means of any gene editing system known in the art. Exemplary gene editing systems include clustered regulatory interspaced short palindromic repeat (CRISPR) system, zinc finger nucleases (ZFNs), and Transcription Activator-Like Effector-based Nucleases (TALEN). ZFNs, TALENs, and CRISPR-based methods are described, e.g., in Gaj et al. Trends Biotechnol. 31.7(2013):397-405; CRISPR methods of gene editing are described, e.g., in Guan et al., Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model. DNA Repair 2016 Jul. 30 [Epub ahead of print]; Zheng et al., Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. BioTechniques, Vol. 57, No. 3, September 2014, pp. 115-124. A person of skill in the art will be aware of these and other options for editing the genome (e.g., knocking out a gene) in a cell, e.g., a production cell.

In some embodiments, eliminating, e.g., knocking out, a copy of a gene encoding an endogenous protein or a regulatory element operably linked to said gene comprises using a CRISPR-Cas9 molecule, e.g., a Cas9 molecule, in combination with a RNA (e.g., gRNA, sgRNA, and/or tracrRNA), specific for the endogenous protein encoding gene or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.

In some embodiments, reducing the level of an endogenous protein in the cell, e.g., production cell, comprises reducing the level of mRNA transcript encoding the endogenous protein, e.g., knockdown, in the cell. In some embodiments, reducing the level of mRNA transcript encoding the endogenous protein, e.g., knockdown, comprises using a siRNA, e.g., capable of binding to nucleic acid, e.g., mRNA, encoding the endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably linked thereto. A person of skill in the art will be aware of tools and techniques for designing and delivering siRNA to a cell. Such tools and/or techniques include, but are not limited to: Dharmacon Horizon siDesign tool, InvivoGen siRNA Wizard, GenScript siRNA Construct Services, IDT Custom Dicer-Substrate siRNA, Sigma-Aldrich siRNA Design Service, or those taught by www.maiweb.com/RNAi/siRNA_Design/. In some embodiments, reducing the level of mRNA transcript encoding the endogenous protein, e.g., knockdown, results in a reduction of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% in the level of mRNA transcript encoding the endogenous protein in the cell.

Products

Provided herein are methods, cells or cell lines, and compositions for producing high yields of a product and/or improving product quality (e.g., increasing the level of product, e.g., recombinant polypeptides, produced) by reducing the level of an endogenous protein in a cell, e.g., production cell. Products described herein include polypeptides, e.g., recombinant proteins, multimeric proteins or complexes; lipid-encapsulated particles, e.g., virus-like particles, vesicles, or exosomes; or other molecules. In an embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. In an embodiment, the product is an exosome. For example, the recombinant polypeptide can be a difficult to express protein or a protein having complex and/or non-natural structures, such as a next generation biologic, e.g., a bispecific antibody molecule, a fusion protein, or a glycosylated protein.

In embodiments, the cell or cell line generated by the methods or compositions described herein produce a product, e.g., a recombinant polypeptide, useful in the treatment of a medical condition, disorder or disease. Examples of medical conditions, disorders or diseases include, but are not limited to, metabolic disease or disorders (e.g., metabolic enzyme deficiencies), endocrine disorders (e.g., hormone deficiencies), dysregulation of hemostasis, thrombosis, hematopoietic disorders, pulmonary disorders, gastro-intestinal disorders, autoimmune diseases, immuno-dysregulation (e g, immunodeficiency), infertility, transplantation, cancer, and infectious diseases.

In embodiments, the product is an exogenous protein, e.g., a protein that is not naturally expressed by the cell. In one embodiment, the protein is from one species while the cell is from a different species. In another embodiment, the protein is a non-naturally occurring protein.

In other embodiments, the product is a protein that is endogenously expressed by the cell. In one embodiment, the product is a protein that is endogenously expressed by the cell at endogenous or natural levels. The present methods and compositions described herein are used to increase the production and quality of the endogenous product, e.g., a naturally occurring product that is naturally produced by the cell. In another embodiment, an exogenous nucleic acid encoding the product, e.g., protein, is introduced to and expressed by the cell. In another embodiment, an exogenous nucleic acid that increases the expression of a product that is endogenously expressed by the cell is introduced into the cell. By way of example, the exogenous nucleic acid comprises a sequence that activates the promoter that controls the expression of an endogenous product of the cell.

The recombinant product can be a therapeutic product or a diagnostic product, e.g., useful for drug screening. The therapeutic or diagnostic product can include, but is not limited to, an antibody molecule, e.g., an antibody or an antibody fragment, a fusion protein, a hormone, a cytokine, a growth factor, an enzyme, a glycoprotein, a lipoprotein, a reporter protein, a therapeutic peptide, or a structural and/or functional fragment or hybrid of any of these. In other embodiments, the therapeutic or diagnostic product is a synthetic polypeptide, e.g., wherein the entire polypeptide or portions thereof is not derived from or has any sequence or structural similarity to any naturally occurring polypeptide, e.g., a naturally occurring polypeptide described above.

In one embodiment, the recombinant product is an antibody molecule. In one embodiment, the recombinant product is a therapeutic antibody molecule. In another embodiment, the recombinant product is a diagnostic antibody molecule, e.g., a monoclonal antibody useful for imaging techniques or diagnostic tests.

An antibody molecule, as used herein, is a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. In an embodiment, the antibody molecule is a full-length antibody or an antibody fragment. Antibodies and multiformat proteins can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. In an embodiment, the antibody is a monoclonal antibody. The antibody may be a human or humanized antibody. In one embodiment, the antibody is an IgA, IgG, IgD, or IgE antibody. In one embodiment, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

“Antibody fragment” refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

In embodiments, the polypeptide is, e.g., BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxins), alglucosidase alpha, daptomycin, YH-16, choriogonadotropin alpha, filgrastim, cetrorelix, interleukin-2, aldesleukin, teceleulin, denileukin diftitox, interferon alpha-n3 (injection), interferon alpha-nl, DL-8234, interferon, Suntory (gamma-1a), interferon gamma, thymosin alpha 1, tasonermin, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, eptoterminalfa, teriparatide, calcitonin, etanercept, hemoglobin glutamer 250 (bovine), drotrecogin alpha, collagenase, carperitide, recombinant human epidermal growth factor, DWP401, darbepoetin alpha, epoetin omega, epoetin beta, epoetin alpha, desirudin, lepirudin, bivalirudin, nonacog alpha, Mononine, eptacog alpha (activated), recombinant Factor VIII+VWF, Recombinate, recombinant Factor VIII, Factor VIII (recombinant), Alphnmate, octocog alpha, Factor VIII, palifermin,Indikinase, tenecteplase, alteplase, pamiteplase, reteplase, nateplase, monteplase, follitropin alpha, rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, iniglucerase, galsulfase, Leucotropin, molgramostim, triptorelin acetate, histrelin (Hydron), deslorelin, histrelin, nafarelin, leuprolide (ATRIGEL), leuprolide (DUROS), goserelin, Eutropin, somatropin, mecasermin, enlfavirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhaled), insulin lispro, insulin deternir, insulin (RapidMist), mecasermin rinfabate, anakinra, celmoleukin, 99 mTc-apcitide, myelopid, Betaseron, glatiramer acetate, Gepon, sargramostim, oprelvekin, human leukocyte-derived alpha interferons, Bilive, insulin (recombinant), recombinant human insulin, insulin aspart, mecasenin, Roferon-A, interferon-alpha 2, Alfaferone, interferon alfacon-1, interferon alpha, Avonex' recombinant human luteinizing hormone, dornase alpha, trafermin, ziconotide, taltirelin, diboterminalfa, atosiban, becaplermin, eptifibatide, Zemaira, CTC-111, Shanvac-B, octreotide, lanreotide, ancestirn, agalsidase beta, agalsidase alpha, laronidase, prezatide copper acetate, rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant human parathyroid hormone (PTH) 1-84, epoetin delta, transgenic antithrombin III, Granditropin, Vitrase, recombinant insulin, interferon-alpha, GEM-215, vapreotide, idursulfase, omnapatrilat, recombinant serum albumin, certolizumab pegol, glucarpidase, human recombinant C1 esterase inhibitor, lanoteplase, recombinant human growth hormone, enfuvirtide, VGV-1, interferon (alpha), lucinactant, aviptadil, icatibant, ecallantide, omiganan, Aurograb, pexigananacetate, ADI-PEG-20, LDI-200, degarelix, cintredelinbesudotox, Favld, MDX-1379, ISAtx-247, liraglutide, teriparatide, tifacogin, AA4500, T4N5 liposome lotion, catumaxomab, DWP413, ART-123, Chrysalin, desmoteplase, amediplase, corifollitropinalpha, TH-9507, teduglutide, Diamyd, DWP-412, growth hormone, recombinant G-CSF, insulin, insulin (Technosphere), insulin (AERx), RGN-303, DiaPep277, interferon beta, interferon alpha-n3, belatacept, transdermal insulin patches, AMG-531, MBP-8298, Xerecept, opebacan, AIDSVAX, GV-1001, LymphoScan, ranpirnase, Lipoxysan, lusupultide, MP52, sipuleucel-T, CTP-37, Insegia, vitespen, human thrombin, thrombin, TransMlD, alfimeprase, Puricase, terlipressin, EUR-1008M, recombinant FGF-I, BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin, SCV-07, OPI-45, Endostatin, Angiostatin, ABT-510, Bowman Birk Inhibitor, XMP-629, 99 mTc-Hynic-Annexin V, kahalalide F, CTCE-9908, teverelix, ozarelix, rornidepsin, BAY-504798, interleukin4, PRX-321, Pepscan, iboctadekin, rhlactoferrin, TRU-015, IL-21, ATN-161, cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DMI, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, MART-1, gp100, tyrosinase, nemifitide, rAAT, CGRP, pegsunercept, thymosinbeta4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-1, AC-100, salmon calcitonin (eligen), examorelin, capromorelin, Cardeva, velafermin, 131I-TM-601, KK-220, T-10, ularitide, depelestat, hematide, Chrysalin, rNAPc2, recombinant Factor V111 (PEGylated liposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, avorelin, ACM-9604, linaclotid eacetate, CETi-1, Hemospan, VAL, fast-acting insulin (injectable, Viadel), insulin (eligen), recombinant methionyl human leptin, pitrakinra, Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn10, talactoferrin, rEV-131, rEV-131, recombinant human insulin, RPI-78M, oprelvekin, CYT-99007 CTLA4-Ig, DTY-001, valategrast, interferon alpha-n3, IRX-3, RDP-58, Tauferon, bile salt stimulated lipase, Merispase, alaline phosphatase, EP-2104R, Melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin, AX-200, SEMAX, ACV-1, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, ALTU-135, recombinant neuraminidase, Vacc-5q, Vacc-4, Tat Toxoid, YSPSL, CHS-13340, PTH(1-34) (Novasome), Ostabolin-C, PTH analog, MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, recombinant plague FIV, AG-702, OxSODrol, rBetV1, Der-p1/Der-p2/Der-p7, PR1 peptide antigen, mutant ras vaccine, HPV-16 E7 lipopeptide vaccine, labyrinthin, WT1-peptide, IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermin, rupintrivir, reticulose, rGRF, HA, alpha-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin, D-4F, ETC-642, APP-018, rhMBL, SCV-07, DRF-7295, ABT-828, ErbB2-specific immunotoxin, DT3SSIL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-B/gastrin-receptor binding peptides, 111In-hEGF, AE-37, trasnizumab-DM1, Antagonist G, IL-12, PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647, L-19 based ra, Re-188-P-2045, AMG-386, DC/1540/KLH, VX-001, AVE-9633, AC-9301, NY-ESO-1 (peptides), NA17.A2 peptides, CBP-501, recombinant human lactoferrin, FX-06, AP-214, WAP-8294A, ACP-HIP, SUN-11031, peptide YY [3-36], FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34, F-18-CCR1, AT-1100, JPD-003, PTH(7-34) (Novasome), duramycin, CAB-2, CTCE-0214, GlycoPEGylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, Factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelix, EP-51216, hGH, OGP-I, sifuvirtide, TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin, r(m)CRP, hepatoselective insulin, subalin, L19-IL-2 fusion protein, elafin, NMK-150, ALTU-139, EN-122004, rhTPO, thrombopoietin receptor agonist, AL-108, AL-208, nerve growth factor antagonists, SLV-317, CGX-1007, INNO-105, teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion, EP-1043, gpE1, gpE2, MF-59, hPTH(1-34), 768974, SYN-101, PGN-0052, aviscumnine, BIM-23190, multi-epitope tyrosinase peptide, enkastim, APC-8024, GI-5005, ACC-001, TTS-CD3, vascular-targeted TNF, desmopressin, onercept, and TP-9201.

In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE™), rituximab (RITUXAN™/MAB THERA™) etanercept (ENBREL™) bevacizumab (AVASTIN™), trastuzumab (HERCEPTIN™), pegrilgrastim (NEULASTA™), or any other suitable polypeptide including biosimilars and biobetters.

Other suitable polypeptides are those listed below and in Table 1 of US2016/0097074:

TABLE 1 Protein Product Reference Usted Drug interferon gamma-1b Actimmune ® alteplase; tissue plasminogen activator Activase ®/Cathflo ® Recombinant antihemophilic factor Advate human albumin Albutein ® Laronidase Aldurazyme ® interferon alfa-N3, human leukocyte Alferon N ® derived human antlhemophilic factor Alphanate ® virus-filtered human coagulation factor AlohaNine ® SD IX Alefacept; recombinant, dimeric fusion Amevive ® protein LFA3-Ig Bivalirudin Angiomax ® darbepoetin alfa Aranesp ™ Bevacizumab Avastin ™ interferon beta-1a; recombinant Avonex ® coagulation factor IX BeneFix ™ Interferon beta-1b Betaseron ® Tositumomab BEXXAR ® antihemophilic factor Bioclate ™ human growth hormone BioTropin ™ botulinum toxin type A BOTOX ® Alemtuzumab Campath ® acritumomab; technetium-99 labeled CEA-Scan ® alglucerase; modified form of beta- Ceredase ® glucocerebrosidase imiglucerase; recombinant form of beta- Cerezyme ® glucocerebrosidase crotalidae polyvalent immune Fab, ovine CroFab ™ digoxin immune fab [ovine] DigiFab ™ Rasburicase Elitek ® Etanercept ENBREL ® epoietin alfa Epogen ® Cetuximab Erbitux ™ algasidase beta Fabrazyme ® Urofollitropin Fertinex ™ follitropin beta Follistim ™ Teriparatide FORTEO ® human somatropin GenoTropin ® Glucagon GlucaGen ® follitropin alfa Gonal-F ® antihemophilic factor Helixate ® Antihemophilic Factor; Factor XIII HEMOFIL adefovir dipivoxil Hepsera ™ Trastuzumab Herceptin ® Insulin Humalog ® antihemophilic factor von Willebrand Humate-P ® factor complex-human Somatotropin Humatrope ® Adalimumab HUMIRA ™ human insulin Humulin ® recombinant human hyaluronidase Hylenex ™ interferon alfacon-1 Infergen ® eptifibatide Integrilin ™ alpha-interferon Intron A ® Palifermin Kepivance Anakinra Kineret ™ antihemophilic factor Kogenate ® FS insulin glargine Lantus ® granulocyte macrophage colony- Leukine ®/ stimulating factor Leukine ®Liquid lutropin alfa for injection Luveris OspA lipoprotein LYMErix ™ Ranibizumab LUCENTIS ® gemtuzumab ozogamicin Mylotarg ™ Galsulfase Naglazyme ™ Nesiritide Natrecor ® Pegfilgrastim Neulasta ™ Oprelvekin Neumega ® Filgrastim Neupogen ® Fanolesomab NeutroSpec ™ (Formerly LeuTech ®) somatropin [rDNA] Norditronin ®/Norditropin Nordiflex ® Mitoxantrone Novantrone ® insulin; zinc suspension; Novolin L ® insulin; isophane suspension Novolin N ® insulin, regular Novolin R ® Insulin Novolin ® coagulation factor VIIa NovoSeven ® Somatropin Nutropin ® immunoglobulin intravenous Octagam ® PEG-L-asparaginase Oncaspar ® abatacept, fully human soluable fusion Orencia ™ protein muromomab-CD3 Orthoclone OKT3 ® high-molecular weight hyaluronan Orthovisc ® human chorionic gonadotropin Ovidrel ® live attenuated Bacillus Calmette-Guerin Pacis ® abatacept, full human soluable fusion Orencia ™ protein muromomab-CD3 Orthoclone OKT3 ® high-molecular weight hyaluronan Orthovisc ® human chorionic gonadotropin Ovidrel ® live attenuated Bacillus Calmette-Guerin Pacis ® peginterferon alfa-2a Pegasys ® pegylated version of interferon alfa-2b PEG-Intron ™ Abarelix (injectable suspension); Plenaxis ™ gonadotropin-releasing hormone antagonist epoietin alfa Procrit ® Aldesleukin Proleukin, IL-2 ® Somatrem Protropin ® dornase alfa Pulmozyme ® Efalizumab; selective, reversible T-cell RAPTIVA ™ blocker combination of ribavirin and alpha Rebetran ™ interferon Interferon beta 1a Rebif ® antihemophilic factor Recombinate ® rAHF/ antihemophilic factor ReFacto ® Lepirudin Refludan ® Infliximab REMICADE ® Abciximab ReoPro ™ Reteplase Retavase ™ Rituxima Rituxan ™ interferon alfa-2^(a) Roferon-A ® Somatropin Saizen ® synthetic porcine secretin SecreFlo ™ Basiliximab Simulect ® Eculizumab SOLIRIS (R) Pegvisomant SOMAVERT ® Palivizumab; recombinantly produced, Synagis ™ humanized mAb thyrotropin alfa Thyrogen ® Tenecteplase TNKase ™ Natalizumab TYSABRI ® human immune globulin intravenous Venoglobulin-S ® 5% and 10% solutions interferon alfa-n1, lymphoblastoid Wellferon ® drotrecogin alfa Xigris ™ Omalizumab; recombinant DNA- Xolair ® derived humanized monoclonal antibody targeting immunoglobulin-E Daclizumab Zenapax ® ibritumomab tiuxetan Zevalin ™ Somatotropin Zorbtive ™ (Serostim ®)

In embodiments, the polypeptide is a hormone, blood clotting/coagulation factor, cytokine/growth factor, antibody molecule, fusion protein, protein vaccine, or peptide as shown in Table 2.

Exemplary recombinant products that can be produced using the methods described herein include, but are not limited to, those provided in the tables below.

TABLE 2 Exemplary Products Therapeutic Product type Product Trade Name Hormone Erythropoietin, Epoein-α Epogen, Procrit Darbepoetin-α Aranesp Growth hormone (GH), Genotropin, Humatrope, Norditropin, somatotropin NovIVitropin, Nutropin, Omnitrope, Protropin, Siazen, Serostim, Valtropin Human follicle-stimulating Gonal-F, Follistim hormone (FSH) Human chorionic Ovidrel gonadotropin Lutropin-α Luveris Glucagon GlcaGen Growth hormone releasing Geref hormone (GHRH) Secretin ChiRhoStim (human peptide), SecreFlo (porcine peptide) Thyroid stimulating hormone Thyrogen (TSH), thyrotropin Blood Factor VIIa NovoSeven Clotting/Coagulation Factor VIII Bioclate, Helixate, Kogenate, Factors Recombinate, ReFacto Factor IX Benefix Antithrombin III (AT-III) Thrombate III Protein C concentrate Ceprotin Cytokine/Growth Type I alpha-interferon Infergen factor Interferon-αn3 (IFNαn3) Alferon N Interferon-β1a (rIFN- β) Avonex, Rebif Interferon-β1b (rIFN- β) Betaseron Interferon-γ1b (IFN γ) Actimmune Aldesleukin (interleukin Proleukin 2(IL2), epidermal theymocyte activating factor; ETAF Palifermin (keratinocyte Kepivance growth factor; KGF) Becaplemin (platelet-derived Regranex growth factor; PDGF) Anakinra (recombinant IL1 Anril, Kineret antagonist) Antibody molecules Bevacizumab (VEGFA mAb) Avastin Cetuximab (EGFR mAb) Erbitux Panitumumab (EGFR mAb) Vectibix Alemtuzumab (CD52 mAb) Campath Rituximab (CD20 chimeric Ab) Rituxan Trastuzumab (HER2/Neu mAb) Herceptin Abatacept (CTLA Ab/Fc fusion) Orencia Adalimumab (TNFα mAb) Humira Etanercept (TNF receptor/Fc Enbrel fusion) Infliximab (TNFα chimeric Remicade mAb) Alefacept (CD2 fusion protein) Amevive Efalizumab (CD11a mAb) Raptiva Natalizumab (integrin α4 Tysabri subunit mAb) Eculizumab (C5mAb) Soliris Muromonab-CD3 Orthoclone, OKT3 Other: Insulin Humulin, Novolin Fusion Hepatitis B surface antigen Engerix, Recombivax HB proteins/Protein (HBsAg) vaccines/Peptides HPV vaccine Gardasil OspA LYMErix Anti-Rhesus(Rh) Rhophylac immunoglobulin G Enfuvirtide Fuzeon Spider silk, e.g., fibrion QMONOS

In embodiments, the protein is a multispecific protein, e.g., a bispecific antibody as shown in Table 3.

TABLE 3 Bispecific Formats Name (other names, Proposed Diseases sponsoring BsAb mechanisms of Development (or healthy organizations) format Targets action stages volunteers) Catumaxomab BsIgG: CD3, Retargeting Approved Malignant (Removab ®, Triomab EpCAM of T cells in EU ascites in Fresenius to tumor, EpCAM Biotech, Trion Fc mediated positive Pharma, effector tumors Neopharm) functions Ertumaxomab BsIgG: CD3, HER2 Retargeting Phase I/II Advanced (Neovii Biotech, Triomab of T cells solid Fresenius to tumor tumors Biotech) Blinatumomab BiTE CD3, CD19 Retargeting Approved Precursor (Blincyto ®, of T cells in USA B-cell AMG 103, to tumor Phase II ALL MT 103, and III ALL MEDI 538, Phase II DLBCL Amgen) Phase I NHL REGN1979 BsAb CD3, CD20 (Regeneron) Solitomab BiTE CD3, Retargeting Phase I Solid tumors (AMG 110, EpCAM of T cells MT110, Amgen) to tumor MEDI 565 BiTE CD3, CEA Retargeting Phase I Gastrointestinal (AMG 211, of T cells adenocancinoma MedImmune, to tumor Amgen) RO6958688 BsAb CD3, CEA (Roche) BAY2010112 BiTE CD3, PSMA Retargeting Phase I Prostate cancer (AMG 212, of T cells Bayer; Amgen) to tumor MGD006 DART CD3, CD123 Retargeting Phase I AML (Macrogenics) of T cells to tumor MGD007 DART CD3, gpA33 Retargeting Phase I Colorectal (Macrogenics) of T cells cancer to tumor MGD011 DART CD19, CD3 (Macrogenics) SCORPION BsAb CD3, CD19 Retargeting (Emergent of T cells Biosolutions, to tumor Trubion) AFM11 TandAb CD3, CD19 Retargeting Phase I NHL and ALL (Affimed of T cells Therapeutics) to tumor AFM12 TandAb CD19, CD16 Retargeting of (Affimed NK cells to Therapeutics) tumor cells AFM13 TandAb CD30, Retargeting of Phase II Hodgkin's (Affimed CD16A NK cells to Lymphoma Therapeutics) tumor cells GD2 (Barbara T cells CD3, GD2 Retargeting Phase I/II Neuroblastoma Ann Karmanos preloaded of T cells and Cancer Institute) with BsAb to tumor osteosarcoma pGD2 (Barbara T cells CD3, Her2 Retargeting Phase II Metastatic Ann Karmanos preloaded of T cells breast cancer Cancer Institute) with BsAb to tumor EGFRBi-armed T cells CD3, EGFR Autologous Phase I Lung and autologous preloaded activated other solid activated T cells with BsAb T cells to tumors (Roger Williams EGFR- Medical Center) positive tumor Anti-EGFR- T cells CD3, EGFR Autologous Phase I Colon and armed activated preloaded activated T pancreatic T-cells (Barbara with BsAb cells to EGFR- cancers Ann Karmanos positive tumor Cancer Institute) rM28 Tandem CD28, Retargeting Phase II Metastatic (University scFv MAPG of T cells melanoma Hospital to tumor Tübingen) IMCgp100 ImmTAC CD3, peptide Retargeting Phase I/II Metastatic (Immunocore) MHC of T cells melanoma to tumor DT2219ARL 2 scFv CD19, CD22 Targeting Phase I B cell leukemia (NCI, University linked to of protein or lymphoma of Minnesota) diphtheria toxin to toxin tumor XmAb5871 BsAb CD19, (Xencor) CD32b NI-1701 BsAb CD47, CD19 (NovImmune) MM-111 BsAb ErbB2, (Merrimack) ErbB3 MM-141 BsAb IGF-1R, (Merrimack) ErbB3 NA (Merus) BsAb HER2, HER3 NA (Merus) BsAb CD3, CLEC12A NA (Merus) BsAb EGFR, HER3 NA (Merus) BsAb PD1, undisclosed NA (Merus) BsAb CD3, undisclosed Duligotuzumab DAF EGFR, Blockade of Phase I Head and neck (MEHD7945A, HER3 2 receptors, and II cancer Genentech, ADCC Phase II Colorectal Roche) cancer LY3164530 Not EGFR, MET Blockade of 2 Phase I Advanced or (Eli Lily) disclosed receptors metastatic cancer MM-111 HSA body HER2, Blockade of 2 Phase II Gastric and (Merrimack HER3 receptors Phase I esophageal Pharmaceuticals) cancers Breast cancer MM-141, IgG-scFv IGF-1R, Blockade of 2 Phase I Advanced (Merrimack HER3 receptors solid tumors Pharmaceuticals) RG7221 CrossMab Ang2, Blockade of 2 Phase I Solid tumors (RO5520985, VEGFA proangiogenics Roche) RG7716 CrossMab Ang2, Blockade of 2 Phase I Wet AMD (Roche) VEGFA proangiogenics OMP-305B83 BsAb DLL4/VEGF (OncoMed) TF2 Dock and CEA, HSG Pretargeting Phase II Colorectal, (Immunomedics) lock tumor for PET breast and or radioimaging lung cancers ABT-981 DVD-Ig IL-1α, IL-1β Blockade of 2 Phase II Osteoarthritis (AbbVie) proinflammatory cytokines ABT-122 DVD-Ig TNF, Blockade of 2 Phase II Rheumatoid (AbbVie) IL-17A proinflammatory arthritis cytokines COVA322 IgG- TNF, IL17A Blockade of 2 Phase I/II Plaque psoriasis fynomer proinflammatory cytokines SAR156597 Tetravalent IL-13, IL-4 Blockade of 2 Phase I Idiopathic (Sanofi) bispecific proinflammatory pulmonary tandem cytokines fibrosis IgG GSK2434735 Dual- IL-13, IL-4 Blockade of 2 Phase I (Healthy (GSK) targeting proinflammatory volunteers) domain cytokines Ozoralizumab Nanobody TNF, HSA Blockade of Phase II Rheumatoid (ATN103, proinflammatory arthritis Ablynx) cytokine, binds to HSA to increase half-life ALX-0761 Nanobody IL-17A/F, Blockade of 2 Phase I (Healthy (Merck HSA proinflammatory volunteers) Serono, cytokines, binds Ablynx) to HSA to increase half-life ALX-0061 Nanobody IL-6R, HSA Blockade of Phase I/II Rheumatoid (AbbVie, proinflammatory arthritis Ablynx; cytokine, binds to HSA to increase half-life ALX-0141 Nanobody RANKL, Blockade of Phase I Postmenopausal (Ablynx, HSA bone resorption, bone loss Eddingpharm) binds to HSA to increase half-life RG6013/ACE910 ART-Ig Factor IXa, Plasma Phase II Hemophilia (Chugai, Roche) factor X coagulation

In embodiments, the product is a polypeptide listed in Table 4.

TABLE 4 Protein Product Reference Listed Drug interferon gamma-1b Actimmune ® alteplase; tissue plasminogen activator Activase ®/Cathflo ® Recombinant antihemophilic factor Advate human albumin Albutein ® Laronidase Aldurazyme ® Interferon alfa-N3, human leukocyte Alferon N ® derived human antihemophilic factor Alphanate ® virus-filtered human coagulation factor IX AlphaNine ® SD Alefacept; recombinant, dimeric fusion Amevive ® protein LFA3-Ig Bivalirudin Angiomax ® darbepoetin alfa Aranesp ™ Bevacizumab Avastin ™ interferon beta-1a; recombinant Avonex ® coagulation factor IX BeneFix ™ Interferon beta-1b Betaseron ® Tositumomab BEXXAR ® antihemophilic factor Bioclate ™ human growth hormone BioTropin ™ botulinum toxin type A BOTOX ® Alemtuzumab Campath ® acritumomab; technetium-99 labeled CEA-Scan ® alglucerase; modified form of beta- Ceredase ® glucocerebrosidase imiglucerase; recombinant form of beta- Cerezyme ® glucocerebrosidase crotalidae polyvalent immune Fab, ovine CroFab ™ digoxin immune fab [ovine] DigiFab ™ Rasburicase Elitek ® Etanercept ENBREL ® epoietin alfa Epogen ® Cetuximab Erbitux ™ algasidase beta Fabrazyme ® Urofollitropin Fertinex ™ follitropin beta Follistim ™ Teriparatide FORTED ® human somatropin GenoTropin ® Glucagon GlucaGen ® follitropin alfa Gonal-F ® antihemophilic factor Helixate ® Antihemophilic Factor; Factor XIII HEMOFIL adefovir dipivoxil Hepsera ™ Trastuzumab Herceptin ® Insulin Humalog ® antihemophilic factor/von Willebrand Humate-P ® factor complex-human Somatotropin Humatrope ® Adalimumab HUMIRA ™ human insulin Humulin ® recombinant human hyaluronidase Hylenex ™ interferon alfacon-1 Infergen ® Eptifibatide Integrilin ™ alpha-interferon Intron A ® Palifermin Kepivance Anakinra Kineret ™ antihemophilic factor Kogenate ® FS insulin glargine Lantus ® granulocyte macrophage colony- Leukine ®/Leukine ® stimulating factor Liquid lutropin alfa for injection Luveris OspA lipoprotein LYMErix ™ Ranibizumab LUCENTIS ® gemtuzumab ozogamicin Mylotarg ™ Galsulfase Naglazyme ™ Nesiritide Natrecor ® Pegfilgrastim Neulasta ™ Oprelvekin Neumega ® Filgrastim Neupogen ® Fanolesomab NeutroSpec ™ (formerly LeuTech ®) somatropin [rDNA] Norditropin ®/ Norditropin Nordiflex ® Mitoxantrone Novantrone ® insulin; zinc suspension; Novolin L ® insulin; isophane suspension Novolin N ® insulin, regular; Novolin R ® Insulin Novolin ® coagulation factor VIIa NovoSeven ® Somatropin Nutropin ® immunoglobulin intravenous Octagam ® PEG-L-asparaginase Oncaspar ® abatacept, fully human soluable Orencia ™ fusion protein muromomab-CD3 Orthoclone OKT3 ® high-molecular weight hyaluronan Orthovisc ® human chorionic gonadotropin Ovidrel ® live attenuated Bacillus Calmette-Guerin Pacis ® peginterferon alfa-2a Pegasys ® pegylated version of interferon alfa-2b PEG-Intron ™ Abarelix (injectable suspension); Plenaxis ™ gonadotropin-releasing hormone Antagonist epoietin alfa Procrit ® Aldesleukin Proleukin, IL-2 ® Somatrem Protropin ® dornase alfa Pulmozyme ® blocker Efalizumab; selective, reversible T-cell RAPTIVA ™ combination of ribavirin and alpha Rebetron ™ interferon Interferon beta 1a Rebif ® antihemophilic factor Recombinate ® rAHF/ antihemophilic factor ReFacto ® Lepirudin Refludan ® Infliximab REMICADE ® Abciximab ReoPro ™ Reteplase Retavase ™ Rituxima Rituxan ™ interferon alfa-2^(a) Roferon-A ® Somatropin Saizen ® synthetic porcine secretin SecreFlo ™ Basiliximab Simulect ® Eculizumab SOLIRIS (R) Pegvisomant SOMAVERT ® Palivizumab; recombinantly produced, humanized mAb Synagis ™ thyrotropin alfa Thyrogen ® Tenecteplase TNKase ™ Natalizumab TYSABRI ® human immune globulin intravenous 5% Venoglobulin-S ® and 10% solutions interferon alfa-n1, lymphoblastoid Wellferon  ® drotrecogin alfa Xigris ™ Omalizumab; recombinant DNA-derived Xolair ® humanized monoclonal antibody targeting immunoglobulin-E Daclizumab Zenapax ® ibritumomab tiuxetan Zevalin ™ Somatotropin Zorbtive ™ (Serostim ®)

In some embodiments, the polypeptide is an antigen expressed by a cancer cell. In some embodiments the recombinant or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is selected from HER2, CD20, 9-O-acetyl-GD3, βhCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carbonic anhydrase IX (MN/CA IX), CCRS, CCR8, CD19, CD22, CD25, CD27, CD30, CD33, CD38, CD44v6, CD63, CD70, CC123, CD138, carcinoma embryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), ErbB2, fetal acetylcholine receptor, fibroblast activation antigen (FAP), fucosyl GM1, GD2, GD3, GM2, ganglioside GD3, Globo H, glycoprotein 100, HER2/neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, LG, Ly-6, melanoma-specific chondroitin-sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC2, MUC3, MUC4, MUC5Ac, MUCSB, MUC7, MUC16, Mullerian inhibitory substance (MIS) receptor type II, plasma cell antigen, poly SA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-alpha precursor, and combinations thereof.

In some embodiments, the polypeptide is an activating receptor and is selected from 2B4 (CD244), α₄β₁ integrin, 132 integrins, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1 (CD66), CRTAM, CS1 (CD319), DNAM-1 (CD226), GITR (TNFRSF18), activating forms of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxicity receptors, NTB-A, PEN-5, and combinations thereof, optionally wherein the (3₂ integrins comprise CD11a-CD 18, CD11 b-CD 18, or CD11c-CD 18, optionally wherein the activating forms of KIR comprise K1R2DS1, KIR2DS4, or KIR-S, and optionally wherein the natural cytotoxicity receptors comprise NKp30, NKp44, NKp46, or NKp80.

In some embodiments, the polypeptide is an inhibitory receptor and is selected from KIR, ILT2/LIR-1/CD85j, inhibitory forms of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, optionally wherein the inhibitory forms of KIR comprise KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

In some embodiments, the polypeptide is an activating receptor and is selected from CD3, CD2 (LFA2, OX34), CDS, CD27 (TNFRSF7), CD28, CD30 (TNFRSF8), CD40L, CD84 (SLAMF5), CD137 (4-1BB), CD226, CD229 (Ly9, SLAMF3), CD244 (2B4, SLAMF4), CD319 (CRACC, BLAME), CD352 (Ly108, NTBA, SLAMF6), CRTAM (CD355), DR3 (TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LTβR (TNFRSF3), OX40 (CD134), NKG2D, SLAM (CD150, SLAMF1), TCRα, TCRβ, TCRδγ, TIM1 (HAVCR, KIM1), and combinations thereof.

In some embodiments, the polypeptide is an inhibitory receptor and is selected from PD-1 (CD279), 2B4 (CD244, SLAMF4), B71 (CD80), B7H1 (CD274, PD-L1), BTLA (CD272), CD160 (BY55, NK28), CD352 (Ly108, NTBA, SLAMF6), CD358 (DR6), CTLA-4 (CD152), LAGS, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2 (TIMD2), TIM3 (HAVCR2, KIM3), and combinations thereof.

Other exemplary proteins include, but are not limited to any protein described in Tables 1-10 of Leader et al., “Protein therapeutics: a summary and pharmacological classification”, Nature Reviews Drug Discovery, 2008, 7:21-39 (incorporated herein by reference); or any conjugate, variant, analog, or functional fragment of the recombinant polypeptides described herein.

Other recombinant protein products include non-antibody scaffolds or alternative protein scaffolds, such as, but not limited to: DARPins, affibodies and adnectins. Such non-antibody scaffolds or alternative protein scaffolds can be engineered to recognize or bind to one or two, or more, e.g., 1, 2, 3, 4, or 5 or more, different targets or antigens.

In one embodiment, the vector comprising a nucleic acid sequence encoding a product, e.g., a polypeptide, e.g., a recombinant polypeptide, described herein further comprises a nucleic acid sequence that encodes a selection marker. In one embodiment, the selectable marker comprises glutamine synthetase (GS); dihydrofolate reductase (DHFR) e.g., an enzyme which confers resistance to methotrexate (MTX); proline, or an antibiotic marker, e.g., an enzyme that confers resistance to an antibiotic such as: hygromycin, neomycin (G418), zeocin, puromycin, or blasticidin. In another embodiment, the selection marker comprises or is compatible with the Selexis selection system (e.g., SUREtechnology Platform™ and Selexis Genetic Elements™, commercially available from Selexis SA) or the Catalant Biologics GPEx® cell line development technology.

In one embodiment, the vector comprising a nucleic acid sequence encoding a recombinant product described herein comprises a selection marker that is useful in identifying a cell or cells comprise the nucleic acid encoding a recombinant product described herein. In another embodiment, the selection marker is useful in identifying a cell or cells that comprise the integration of the nucleic acid sequence encoding the recombinant product into the genome, as described herein. The identification of a cell or cells that have integrated the nucleic acid sequence encoding the recombinant protein can be useful for the selection and engineering of a cell or cell line that stably expresses the product.

In one embodiment, the product differs from a polypeptide from Tables 1-4 at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues. In another embodiment, the product differs from a polypeptide from Table 2 or 3 at no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% of its amino acid residues. Methods for determining percent identity are discussed above.

Other recombinant products include non-antibody scaffolds or alternative protein scaffolds, such as, but not limited to: DARPins, affibodies and adnectins.

Other exemplary therapeutic or diagnostic proteins include, but are not limited to any protein described in Tables 1-10 of Leader et al., “Protein therapeutics: a summary and pharmacological classification”, Nature Reviews Drug Discovery, 2008, 7:21-39 and as described in Walsh, “Biopharmaceutical benchmarks 2014”, Nature Biotechnology, 2014, 32:992-1000 (each incorporated herein by reference); or any conjugate, variant, analog, or functional fragment of the recombinant polypeptides described herein.

Production Applications

The methods and cells or cell lines disclosed herein can be used to produce a variety of products, evaluate various cell lines, or to evaluate the production of various cell lines for use in a bioreactor or processing vessel or tank, or, more generally with any feed source. The devices, facilities and methods described herein are suitable for culturing any desired cell line including prokaryotic and/or eukaryotic cell lines. Further, in embodiments, the devices, facilities and methods are suitable for culturing suspension cells or anchorage-dependent (adherent) cells and are suitable for production operations configured for production of pharmaceutical and biopharmaceutical products—such as polypeptide products or cells and/or viruses such as those used in cellular and/or viral therapies.

As mentioned, in embodiments, devices, facilities and methods allow for the production of eukaryotic cells, e.g., mammalian cells or lower eukaryotic cells such as for example yeast cells or filamentous fungi cells, or prokaryotic cells such as Gram-positive or Gram-negative cells and/or products of the eukaryotic or prokaryotic cells, e.g., proteins, peptides, antibiotics, amino acids, synthesised by the eukaryotic cells in a large-scale manner Unless stated otherwise herein, the devices, facilities, and methods can include any desired volume or production capacity including but not limited to bench-scale, pilot-scale, and full production scale capacities.

Moreover and unless stated otherwise herein, the devices, facilities, and methods can include any suitable reactor(s) including but not limited to stirred tank, airlift, fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and/or spouted bed bioreactors. As used herein, “reactor” or “bioreactor” can include a fermenter or fermentation unit, or any other reaction vessel and the terms “reactor” and “bioreactor” are used interchangeably with “fermenter.” For example, in some aspects, a bioreactor unit can perform one or more, or all, of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO₂ levels, maintenance of pH level, agitation (e.g., stirring), and/or cleaning/sterilizing. Example reactor units, such as a fermentation unit, may contain multiple reactors within the unit, for example the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors in each unit and/or a facility may contain multiple units having a single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi fed-batch, fed-batch, perfusion, and/or a continuous fermentation processes. Any suitable reactor diameter can be used. In embodiments, the bioreactor can have a volume between about 100 mL and about 50,000 L. Non-limiting examples include a volume of 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Additionally, suitable reactors can be multi-use, single-use, disposable, or non-disposable and can be formed of any suitable material including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and/or glass. In some embodiments, suitable reactors can be round, e.g., cylindrical. In some embodiments, suitable reactors can be square, e.g., rectangular. Square reactors may in some cases provide benefits over round reactors such as ease of use (e.g., loading and setup by skilled persons), greater mixing and homogeneity of reactor contents, and lower floor footprint.

In embodiments and unless stated otherwise herein, the devices, facilities, and methods described herein for use with methods of making a preparation can also include any suitable unit operation and/or equipment not otherwise mentioned, such as operations and/or equipment for separation, purification, and isolation of such products. Any suitable facility and environment can be used, such as traditional stick-built facilities, modular, mobile and temporary facilities, or any other suitable construction, facility, and/or layout. For example, in some embodiments modular clean-rooms can be used. Additionally and unless otherwise stated, the devices, systems, and methods described herein can be housed and/or performed in a single location or facility or alternatively be housed and/or performed at separate or multiple locations and/or facilities.

By way of non-limiting examples and without limitation, U.S. Publication Nos. 2013/0280797; 2012/0077429; 2011/0280797; 2009/0305626; and U.S. Pat. Nos. 8,298,054; 7,629,167; and 5,656,491, which are hereby incorporated by reference in their entirety, describe example facilities, equipment, and/or systems that may be suitable.

Bioreactor Setup and Conditions

In one aspect, a bioreactor of the disclosure or a bioreactor utilized by a method of the disclosure is a single-use bioreactor.

The single-use bioreactor may include a bioprocess container, a shell, at least one agitator, at least one sparger, at least one gas filter inlet port for the sparger(s) and headspace overlay, at least one fill port, at least one harvest port, at least one sample port, and at least one probe.

In one embodiment, the present disclosure is directed to a bioreactor comprising a bioprocess container. The bioprocess container is made from a liquid impermeable and flexible shape-conforming material. For instance, the bioprocess container can be made from a flexible film, such as a multi-layer film. In one embodiment, for instance, the film is comprised of a polyethylene polymer, such as a low density polyethylene that has been modified to form a hydrophilic surface. The hydrophilic surface is for contact with cell cultures within the bioreactor and improves wettability. In one embodiment, the polyethylene polymer is modified by being subjected to irradiation, photo or plasma induction, or oxidation.

The bioprocess container can have a top, a bottom, and at least one side wall therebetween. The bioprocess chamber can define a hollow enclosure for receiving a culture media. The hollow enclosure can have any suitable volume, such as 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters.

The bioreactor can include at least one inlet port for feeding materials into the hollow enclosure of the bioprocess container. A mixing device comprising a rotatable shaft coupled to at least one agitator can extend into the hollow enclosure of the bioprocess container. In one embodiment, the rotatable shaft can be collapsible. For instance, the rotatable shaft can include at least one impeller made from a hydrophilic polymer material that is collapsible or foldable towards the rotatable shaft.

The bioreactor can also include at least one baffle configured to extend adjacent to the side wall of the bioprocess container in a longitudinal direction. The baffle can have a shape that extends radially inward from the side wall an amount sufficient to affect fluid flow in the hollow enclosure during mixing of a culture media by the mixing device. The baffle can be collapsible and/or foldable. In one embodiment, for instance, the baffle can define an inflatable fluid bladder making the baffle capable of being inflated and deflated. The baffle can be integral with the bioprocess container meaning that the baffle is formed into the flexible shape-forming material. Alternatively, the baffle can be separate from the bioprocess container. The baffle can be configured to be placed inside the hollow enclosure or can be placed outside the hollow enclosure. When placed outside the hollow enclosure, the side wall of the bioprocess container conforms around the shape of the baffle. For example, in one embodiment, the baffle can be removably attached to an outer metallic shell. The bioprocess container can be placed in the metallic shell for conforming around the shape of the baffle. The bioreactor, in one embodiment, can include from about two to about six baffles that are spaced around a circumference of the hollow enclosure of the bioprocess container.

In one embodiment, the bioprocess container has a diameter and the one or more baffles extend radially inward a distance of from about 3% to about 20%, such as from about 5% to about 15% of the diameter of the bioprocess container.

The bioreactor can further include at least one sparger. The sparger, for instance, may comprise a ballast sparger that comprises a gas tube having a longitudinal portion and a lateral portion. The longitudinal portion can extend vertically into the hollow enclosure of the bioprocess container. The lateral portion, on the other hand, can be located at an end of the longitudinal portion below the agitator. The lateral portion can define a plurality of holes for releasing a gas into a culture media contained within the bioprocess container. In one embodiment, the plurality of holes are drilled. The lateral portion can have any suitable shape. In one embodiment, the lateral portion can be configured to engage the rotatable shaft of the mixing device for stabilizing the shaft. The rotatable shaft may extend through the lateral portion or can be housed within a shaft receiving member formed into the lateral portion.

In one embodiment, the bioreactor includes a first subsurface sparger and a second supersurface sparger. The plurality of holes in the subsurface sparger can be larger or smaller than the plurality of holes on the supersurface sparger. In one embodiment, the plurality of holes are drilled.

In one embodiment, the bioreactor can include at least one feed line that extends into the hollow enclosure for feeding fluids into the bioprocess container. The feed line can include a subsurface fluid outlet positioned adjacent the agitator. The fluid outlet can be associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction. For instance, the fluid control device may comprise a one-way valve.

In another embodiment, the bioreactor can include a feed line positioned at the top of the bioprocess container. The feed line can include a supersurface fluid discharge positioned above a volume of culture media residing in the bioprocess container. The supersurface fluid discharge can be located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container. In one embodiment, the agitator can form a circumference when rotated and the supersurface fluid discharge of the feed line can be positioned above the circumference of the agitator such that fluids flowing through the fluid discharge contact the culture media within the circumference.

The bioreactor can be placed in operative association with a load cell for indicating a mass of a culture media contained within the hollow enclosure. The bottom of the bioprocess container can have a dome-shape for facilitating drainage. For instance, the bioprocess container can include a drain line located at the bottom of the bioprocess container. A fluid collecting device can be positioned in-between the hollow enclosure of the bioprocess container and the drain line. The fluid collecting device can have a shape configured to induce a vortex flow of fluids from the bioprocess container into the drain line. In one embodiment, the drain line has a cross-sectional area that is proportional to the volume of the hollow enclosure. For example, for exemplary purposes, the drain line can have a cross-sectional area of from about 0.3 mm² to about 0.7 mm², such as from about 0.4 mm² to about 0.6 mm² per liter of volume of the hollow enclosure.

In one embodiment, the bioprocess container can include a plurality of ports for connecting to a plurality of supply lines for feeding fluids to the bioprocess container. Each port and corresponding supply line can include matching indicators for assisting a user in connecting the supply lines to the respective ports. The matching indicators, for instance, may comprise color such that each port and corresponding supply line are color-coded. Matching indicia can also be applied to feed lines and any corresponding ports and to spargers and any corresponding connectors.

In one embodiment, the bioprocess container can include ports that comprise universal connectors. The ports can have a first end and a second end. The first end can be for forming a reconnectable attachment to a respective supply line. Each supply line can include a fluid filter positioned upstream from the corresponding ports.

The present disclosure is also directed to a bioreactor system. The bioreactor system can include a bioprocess container made from a liquid impermeable and flexible shape-conforming material. The bioprocess container can have a top, a bottom, and at least one side wall therebetween. The bioprocess chamber can define a hollow enclosure for receiving a culture media. The bioprocess container can also include a plurality of inlet ports for feeding materials into the hollow enclosure. A drain line can be positioned at the bottom of the bioprocess container for draining fluids. A mixing device can extend into the hollow enclosure of the bioprocess container and can comprise a rotatable shaft coupled to at least one agitator.

The bioreactor system can further include at least one sensor in operative association with the bioprocess container for monitoring at least one parameter within the hollow enclosure. The at least one sensor can comprise a pH sensor, a dissolved carbon dioxide sensor, a dissolved oxygen sensor, redox sensor, a load cell, a temperature sensor, or a tachometer. A controller can be placed in communication with the at least one sensor. The controller can be configured to receive information from the at least one sensor and, based on the information, to control a fluid supply for varying a flow rate of a fluid from the fluid supply into the hollow enclosure of the bioprocess container for maintaining the at least one parameter of a culture media contained within the hollow enclosure within preset limits.

For example, in one embodiment, the bioreactor system can include a carbon dioxide gas supply in fluid communication with the bioprocess container and a liquid alkali supply also in fluid communication with the bioprocess container. The at least one sensor can comprise a pH sensor and the controller can be configured to regulate pH levels of a culture media within the preset limits by adding amounts of carbon dioxide gas from the carbon dioxide gas supply for selectively lowering the pH or by adding amounts of an alkali from the liquid alkali supply for selectively increasing the pH. In one embodiment, the system can include a first pH sensor and a second pH sensor both in communication with the controller.

In yet another embodiment, the bioreactor system can include an oxygen gas supply and the at least one sensor can comprise a dissolved oxygen sensor. The controller can regulate dissolved oxygen levels within a culture media within preset limits by periodically adding amounts of oxygen gas from the oxygen gas supply to a culture media based on information received from the dissolved oxygen sensor.

In still another embodiment, the bioreactor system can include a carbon dioxide gas supply and wherein the at least one sensor comprises a dissolved carbon dioxide sensor. The controller can be configured to regulate dissolved carbon dioxide levels within a culture media within preset limits by periodically adding amounts of carbon dioxide gas from the carbon dioxide gas supply to a culture media based upon information received from the dissolved carbon dioxide sensor.

In still another embodiment, the bioreactor system can include a thermal jacket surrounding the bioprocess container. The thermal jacket can be in fluid communication with at least one of a heated fluid or a chilled fluid. The bioreactor system can further include a temperature sensor for sensing a temperature of a culture media contained within the bioprocess container. The temperature sensor can be in communication with the controller. The controller can be configured to receive information from the temperature sensor, and, based on the information, control flow of a fluid into the thermal jacket for increasing or decreasing the temperature of a culture media contained in the bioprocess container for maintaining a culture media within preset temperature limits.

In another embodiment, the bioreactor system can further include a tachometer for monitoring a rotational speed of the rotatable shaft coupled to the at least one agitator. The tachometer can be in communication with the controller. The controller can be in communication with a motor that rotates the shaft. The controller can be configured to control the motor in a manner that rotates the shaft at a predetermined speed based upon information received from the tachometer.

The controller may comprise one or more microprocessors.

In one embodiment, the controller can be configured to receive information from multiple sensors in order to control multiple parameters within the bioreactor.

In one embodiment, one or more of the sensors described above can be integrated into the bioprocess container and can be disposable with the bioprocess container.

The present disclosure is also directed to a bioreactor comprising a bioprocess container made from a liquid impermeable and flexible shape-conforming material. The bioprocess container can have a top, a bottom, and at least one side wall therebetween. The bioprocess chamber can define a hollow enclosure for receiving a culture media. At least one feed line can extend into the hollow enclosure for feeding a fluid into the bioprocess container.

In one embodiment, the feed line includes a subsurface fluid outlet positioned adjacent to an agitator. The fluid outlet can be associated with a fluid control device that only permits fluid to flow out of the fluid outlet and prevents fluid flow in an opposite direction.

In an alternative embodiment, the feed line can comprise a supersurface fluid discharge positioned above a volume of a culture media residing in the bioprocess container. The supersurface fluid discharge can be located such that a fluid flowing through the fluid discharge makes direct contact with a culture media contained within the bioprocess container without contacting the side wall.

In one embodiment, the bioreactor can include a first feed line that includes the subsurface fluid outlet and a second feed line including the supersurface fluid discharge. In one embodiment, the bioreactor can contain from about one to about five, such as from about two to about three feed lines that have a supersurface fluid discharge.

In yet another embodiment, the present disclosure is directed to a method for producing a single use bioreactor. The method includes the steps of constructing a bioprocess container from a liquid impermeable and flexible shape-conforming material. The bioprocess container having a top, a bottom, and at least one side wall therebetween. The bioprocess chamber defines a hollow enclosure for receiving a culture media. The hollow enclosure can have a volume of from about of 1-250 milliliters, 250 milliliters to 50 liters, 50 to 800 liters, or 800-200,000 liters. The bioprocess container includes a plurality of inlet ports for feeding materials into the hollow enclosure of the bioprocess container. Each inlet port has a diameter.

A mixing device is inserted into the hollow enclosure. The mixing device comprises a rotatable shaft coupled to at least one agitator. At least one sparger is also inserted into the hollow enclosure of the bioprocess container. The sparger comprises a gas tube that has a longitudinal portion and a lateral portion. The longitudinal portion extends vertically into the hollow enclosure. The lateral portion is located at an end of the longitudinal portion below the agitator. The lateral portion defines a plurality of holes for releasing a gas into a culture media contained within the bioprocess container. The plurality of holes have a diameter.

A drain line is connected to the bottom of the bioprocess container. The drain line has a cross-sectional area.

In accordance with the present disclosure, the diameter of the inlet ports, the diameter of the plurality of holes on the sparger, and the cross-sectional area of the drain line are proportional to the volume of the hollow enclosure. The drain line, for instance, can have a cross-sectional area of from about 0.3 mm² to about 0.7 mm² per liter of volume of the hollow enclosure.

The present disclosure is also directed to a bioreactor comprising a bioprocess container made from a liquid impermeable and flexible shape-conforming material. The bioprocess chamber can define a hollow enclosure for receiving a culture media and can include at least one inlet port. A mixing device comprising a rotatable shaft coupled to a plurality of agitators can extend into the hollow enclosure of the bioprocess container.

In accordance with the present disclosure, the bioreactor can further include a [ ]cell retention chamber in fluid communication with the hollow enclosure of the bioprocess container. A filtrate outlet can be placed in fluid communication with the cell retention chamber. The filtrate outlet includes a biofilter that is permeable to liquids but impermeable to biological materials contained in a culture media. The filtrate outlet is for removing liquids from the cell retention chamber continuously or periodically. A flow regulator is configured to alternate flow of a culture media between the hollow enclosure of the bioprocess container and the cell retention chamber for carrying out a perfusion process.

The flow regulator, for instance, can be in communication with a pressurized gas source and a vacuum source. The flow regulator can be configured to alternatively apply a vacuum or a gas pressure to a fluid contained in the cell retention chamber for recycling fluids back and forth between the hollow enclosure of the bioprocess container and the cell retention chamber.

In one embodiment, the flow regulator can include a reciprocating diaphragm that alternates between applying pressure and applying a suction force to the fluid contained in the cell retention chamber.

The present disclosure is also directed to a bioreactor comprising a bioprocess container made from a liquid impermeable and flexible shape-conforming material. The bioprocess container defines a hollow enclosure for receiving a culture media. A mixing device comprising a rotatable shaft coupled to at least one agitator can extend into the hollow enclosure of the bioprocess container. In accordance with the present disclosure, the agitator can be collapsible onto the rotating shaft. For instance, the agitator can comprise an impeller comprising at least one blade element. The blade element can be foldable towards the rotatable shaft. In one embodiment, the rotatable shaft is coupled to a first impeller and a second impeller and both impellers can include at least one blade element that is foldable. A retaining ring can be positioned on the shaft. The retaining ring can include an agitator engaging position and an agitator disengaging position for holding the agitator in an upright position during mixing or in a collapsed and folded position respectively.

In one embodiment, the rotatable shaft comprises a metallic reinforcing rod surrounded by a shaft sleeve. The metallic reinforcing rod, which can be made from stainless steel, can be made from multiple pieces that are attached together. The top of the reinforcing rod can include a magnetic member for magnetically engaging a motor. The shaft sleeve can be comprised of a polymeric material. The agitator on the shaft can also be made from a polymeric material, such as a hydrophilic polymer. For example, the shaft sleeve and the agitator can comprise a polyethylene polymer that has been modified by being subjected to irradiation, photo or plasma induction, or oxidation.

In one embodiment, a single use bioreactor system according to the instant disclosure comprises: a single use cell culture bioprocess container (“SUB”), a reusable shell in which the SUB is held during operation, and a controller that controls the operation of the SUB and associated sub-systems and processes. Associated sub-systems include an agitation system, a baffle system, a sparger system, a feeding system, a harvesting system, a monitoring system, control system(s), and a fill system.

In one embodiment, each of cell culture contacting and process fluid contacting surface of the SUB are preferably animal derived component free.

According to one aspect of the disclosure, a single-use bioreactor is provided. The single-use bioreactor may include a bioprocess container, a shell, at least one agitator, at least one sparger, at least one gas filter inlet port for the sparger(s) and headspace overlay, at least one fill port, at least one harvest port, at least one sample port, and at least one probe.

A single use bioreactor of the disclosure may also be used with a single use bioreactor (SUB) system. The system may include a flexible bioreactor bioprocess container that is for single use and disposable, a SUB shell configured to hold the flexible bioreactor bioprocess container, an agitator, a sparger, a plurality of ports, and at least one controller configured to control a plurality of parameters associated with the SUB system such that the SUB system produces biomaterial corresponding to biomaterial capable of being produced in a similarly sized stainless steel bioreactor.

In accordance with the present disclosure, the rotatable shaft can be coupled to a top impeller and to a bottom impeller. Both the top impeller and the bottom impeller can be made from a polymer material. For instance, in one embodiment, the impellers may be 3-D printed. The top impeller and the bottom impeller can both define a hydrophilic surface. For instance, the polymer material used to form the impellers can comprise a hydrophilic polymer or can comprise a polymer that has been surface modified so as to render the surface hydrophilic.

In one embodiment, for instance, the top impeller and bottom impeller are made from a polyolefin polymer, such as polyethylene or polypropylene. In one embodiment, low density polyethylene can be used. The low density polyethylene can be modified by being subjected to irradiation, photo or plasma induction, or oxidation to form a hydrophilic surface.

The top impeller can comprise a hydrofoil impeller. The bottom impeller, on the other hand, can comprise a four pitched-bladed high solidity impeller. The impeller to tank diameter ratio can be from about 0.35 to about 0.55, such as from about 0.44 to about 0.46. The top impeller and the bottom impeller can have power numbers (N_(e)) of from about 0.1 to about 0.9 and can have flow numbers (N_(q)) of from about 0.4 to about 0.9.

In some embodiments, a bioreactor of the disclosure is a bioreactor as described in U.S. Pat. No. 9,670,446, the content of which is hereby incorporated by reference in its entirety. In some embodiments, a bioreactor of the disclosure comprises a part or feature of a bioreactor described in U.S. Pat. No. 9,670,446.

In one embodiment, a bioreactor can be operably coupled to a harvest vessel, e.g., harvest vessel. The harvest vessel may comprise a means to mix culture and gas (e.g., to aerate culture), e.g., to ensure adequate oxygenation of culture. In an embodiment, the harvest vessel comprises a supernatant having been deposited in the harvest vessel, e.g., from the bioreactor), and a head space comprising gas, e.g., air, oxygen, or a mixture of air and oxygen. In an embodiment, the harvest vessel uses surface aeration to oxygenate culture supernatent with the headspace gas e.g. air or oxygen or air and oxygen gas mixture. Surface aeration involves cascading or flowing supernatant from the J-tube inlet port down the harvest vessel wall and from the supernatant liquid surface in contact with the headspace In some embodiments, travel or cascade results in an increase in oxygenation level of the culture.

The harvest vessel may include at least one mixer, at least one gas filter inlet ports for the headspace overlay, at least one fill port with an internal J-tube directed towards the vessel wall, at least one harvest port, at least one sample port, at least one temperature probe, at least one redox probe, at least one DOT probe, and at least one pH probe, at least one temperature control, at least one gas flow, e.g., at least one air flow control and at least one O2 flow control.

In one embodiment, the harvest vessel comprises a head space gas mixture of air/O2 that maintains a DOT in a range greater than 40% air saturation to less than or equal to 500% air saturation

In an embodiment, a bioreactor or harvest vessel comprises a redox probe, e.g., an in-line redox probe, e.g., a Mettler Toledo in-line redox probe.

In some embodiments, a method comprises a bioreactor or harvest vessel is capable of providing, e.g., adding, or regulating, e.g., increasing or decreasing activity/abundance of, components of the thioredoxin or glutathione/glutaredoxin systems to provide or maintain a redox potential in a culture or cells of a culture or culture supernatent. The thioredoxin and glutathione/glutaredoxin systems and their components are known in the art. See, e.g., Holmgren et al. Biochem Soc Trans. 2005 December; 33(Pt 6):1375-7; Nordberg and Arner. Free Radic Biol Med. 2001 Dec. 1; 31(11):1287-312; Ghezzi P. Biochem Soc Trans. 2005 December; 33(Pt 6):1378-81; Ivarsson et al. Diabetes. 2005 July; 54(7):2132-42; Sen, C K. Biochem Pharmacol 55 (11), 1747-1758. 1998 Jun. 1; and May et al. J Biol Chem. 1997 Sep. 5; 272(36):22607-10, the contents of which are hereby incorporated by reference in their entirety.

In an embodiment, a method comprises or a bioreactor or harvest vessel is capable of providing, e.g., adding, or regulating, e.g., increasing or decreasing activity/abundance of, GILT (gamma-interferon-inducible lysosomal thiol reductase). See, e.g., Rausch and Hastings. Mol Immunol. 2015 December; 68(2 Pt A):124-8. doi: 10.1016/j.molimm 2015.06.008. Epub 2015 Jun. 23; and Hastings and Cresswell. Antioxid Redox Signal. 2011 Aug. 1; 15(3): 657-668, the contents of which are hereby incorporated by reference in their entirety. In an embodiment, a method comprises or a bioreactor or harvest vessel is capable of providing, e.g., adding, ascorbic acid, dehydroascorbic acid, or an ascorbic acid or dehydroascorbic acid modifying component to the growth culture. The redox potential regulating properties of ascorbic acid and dehydroascorbic acid are known in the art. See, e.g., Winkler et al. Free Radic Biol Med. 1994 October; 17(4):333-49, which is hereby incorporated by reference in its entirety.

In an embodiment, a method comprises or a bioreactor is capable of providing intracellular agents, e.g., adding redox potential-indicating labels, e.g., redox-sensitive dyes or molecular probes, to the production culture. Such redox potential-indicating labels may be useful for monitoring the redox potential of the culture or cells within the culture. Redox potential-indicating labels include, but are not limited to: 2,2′-bipyridine (Ru complex), Nitrophenanthroline (Fe complex), N-Phenylanthranilic acid, 1,10-Phenanthroline iron(II) sulfate complex (Ferroin), N-Ethoxychrysoidine, 2,2′-Bipyridine (Fe complex), 5,6-Dimethylphenanthroline (Fe complex), o-Dianisidine, Sodium diphenylamine sulfonate, Diphenylbenzidine, Diphenylamine, Viologen, Sodium 2,6-Dibromophenol-indophenol, Sodium 2,6-Dichlorophenol-indophenol, Sodium o-Cresol indophenol, Thionine (Lauth's violet), Methylene blue, Indigotetrasulfonic acid, Indigotrisulfonic acid, Indigo carmine (Indigodisulfonic acid), Indigomono sulfonic acid, Phenosafranin, Safranin, Neutral red, labels disclosed in any document incorporated herein, and labels disclosed in Schwarzlander M et al, Antioxid Redox Signal. 2016 May 1; 24(13):680-712, which is hereby incorporated by reference in its entirety.

In another embodiment, a method comprises or a bioreactor or harvest vessel is capable of providing extracellular agents, e.g., metabolites, transition state metal ion as described herein, to the production culture.

In some embodiments, a bioreactor or bioreactor for use in a method of the disclosure can be set up according to the following principles:

-   -   The functional set up of the bioreactor while the production         cultures is harvested is important in ensuring the culture         exiting the bioreactor is well oxygenated and carries dissolved         oxygen into the subsequent processing steps. Once cell culture         has reached the target temperature to initiate the harvest step         ensure all process controls that are likely to antagonize the         oxygenation of the production culture are inhibited. These may         include inhibition of sparged nitrogen gas flow if active,         inhibition of on demand CO₂ gas flow control if active,         inhibition of on demand alkali control if active, and inhibition         of feed application if active.     -   The nitrogen sparge gas flow is normally used as a ballast to         control dissolved oxygen tension (DOT) to a set point when the         cellular demand for oxygen is low, however this may be kept         actively flowing during the course of the production stage.         Nitrogen is also used in airlift bioreactors as carrier ballast         to maintaining mixing in these vessels. However where nitrogen         sparge gas is used it must be inhibited to prevent diluting the         oxygen composition of sparged gases used for aerating the         production culture during the harvest step as this will prevent         the dissolved oxygen, DOT in the culture from reaching 100% air         saturation (if air only sparge is used) or the maximum DOT         expected (if a blend of air and oxygen is used). Sparged         nitrogen therefore limits the maximum dissolved oxygen         concentration expected for a given sparge of air alone or sparge         of air-oxygen mixture. The CO₂ gas flow is sparged into the         production culture in response to process pH rising above         control range. When the CO₂ gas flow sparge is active it also         dilutes the oxygen composition of the sparge gas which in turn         limits the maximum dissolved oxygen concentration expected for a         given sparge of air alone or sparge of air-oxygen mixture.         Therefore its inactivation during the harvesting of production         culture prevents the dilution of the aerating sparge gas.     -   Addition of alkali solution to the production culture in         response to the process pH falling below the control range is         needed for tight pH control during the cultivation of the         production culture. However there are potential risks of greater         cell damage and cell death with application of alkali solution         while the production culture is being harvested due to a         continually decrease operating volume and overdosing of the         alkali solution. The application of feeds into the production         culture is necessary while the culture is actively growing and         metabolizing. However during the harvest the continually         decreasing operating volume impacts the mixing behavior of the         bioreactor and result in greater cell damage and cell death with         application of feed solution due to the non-physiological nature         of feeds (high osmolality and high or low pH) and         micro-environments created in poorly mixed vessels.     -   Once production culture has reached the target temperature to         initiate the harvest step ensure all process controls that are         likely to promote the oxygenation of the production culture are         activated. These may include activation of sparged air and/or         oxygen gas flow if not active, activation of head-space air         and/or oxygen flow, if not active, and finally activation of         head-space pressure if not active. The on-demand air and or         oxygen sparge flow may become very low or even stop once the         production culture has stopped growing, or when it is cooled         down, in readiness for harvest. The continued application of air         and oxygen sparge at various continuous fixed flow rates is         critical to oxygenating the production culture and in ensuring         the production culture carries dissolved oxygen into the cell         clarifying filter housings or centrifuge and flow paths between         the bioreactor and filter housing or centrifuge and between the         filter housing or centrifuge and supernatant collection vessel         (e.g., harvest vessel). The head-space or overlay aeration is         typically active during the cultivation of the production         culture and ensures bioreactor head-space is continually purged         of metabolic CO₂ gas. The continued overlay aeration of the         production culture during harvest promotes surface oxygenation         of the culture albeit more slowly than achieved with sparge         gases but avoid production of foam while the production culture         is draining out of the bioreactor.     -   For bioreactors capable of operating with head-space pressure         and where pressure is used during the cultivation of production         culture to aid greater solubility of gases into the liquid phase         (culture) or to maintain a positive pressure within the         bioreactor to prevent ingress of environmental contaminants         across the vessel's sterile boundaries it is recommended that         this is maintained during the harvest. In practice where the         harvest is driven by pressure the culture oxygenation will be         aided provided the gas used to pressurize the bioreactor is air         or a mixed blend of air and oxygen. However if the harvest is         driven by a pump, head-space pressure is not typically used. The         use of head-space pressure, with an air or air/oxygen gas         mixture, in such circumstances is beneficial in aiding culture         oxygenation and resisting the harvest tubing from collapsing         under the suction head upstream of the pump head when culture is         flowing at required fast rates dependent on scale of operation         needed during the harvesting operation.     -   For single use bioreactors the design of the harvest line with         respect to internal bore size and wall strength to resist         collapsing under suction produced upstream of the pump head at         high flow rates is important in ensuring the harvest flow rate         is not impeded through tube occlusion. The impact of tube         occlusion is not only in restricting the harvest flow rate but         may promote greater cell death and cell lysis and degassing of         dissolved oxygen from the culture. The tube occlusion impacts         have the potential to expose the cells and product to         hypoxic/anoxia environment with the filter housing through         increase the residence time in the housing at slower flow rate,         promote the release of intracellular factors through greater         cell death and cell lysis passing through a constricted opening         and remove the dissolved oxygen from the culture by degassing         within the suction zone and so lower amounts of the dissolved         oxygen carried by the culture into the filter housings. In one         embodiment, the methods disclosed herein avoid these negative         impact on product stability.

The internal diameter of the harvest tubing and choice of pump used are selected to attain the required flow rate to achieve the process volumetric throughput (Litres of process supernatant per filter area, L/m²) for the cell clarification step by depth filters or centrifuge coupled to depth filters. The material of construction of the harvest line needs to have the rigidity to resist collapsing in the suction head produced upstream of the pump head. It is proposed materials other the commonly-used platinum-cured silicon or C-flex be used in the construction of the harvest tubing. The use of braided tubing may be considered as an alternative to the commonly used materials.

-   -   In some embodiments, a method comprises or a bioreactor is         capable of optimization of depth filter area to avoid fouling         and blockage of the filters during the filter clarification step         and thereby minimizing the residence time of the cell culture         within the filter housings is integral step in ensuring the         oxygenation of the production culture and the dissolved oxygen         it carries out of the bioreactor is not exhausted while the         culture flows into and resides in the filter housings before         passing out as supernatant. Furthermore, the flow rate should be         adequate to minimize the residence time of the supernatant         between the clarification step (filter housing) and the c         supernatant collection vessel (e.g., harvest vessel).

The cell clarification filter (primary, secondary and tertiary) areas are optimized to ensure the required process volumetric throughput is met without loss of flux (L/m2/h) or filtrate quality (through breakthrough of particulate) across the filters to the extent that it impacts the performance of secondary or Stage 2 and tertiary or Stage 3 filters downstream of the primary or Stage 1 filters. Additionally, the filter area and filter types selected for optimal cell clarification must avoid higher pressure differentials between the different filter stages which signifies filter fouling and imminent filter blockage. It is therefore expected that a decrease in the flux across the different serially-coupled filters would lead to increased residence time within the filter housings and leading to greater consumption/exhaustion of dissolved oxygen in the supernatant within the filter housing leading to hypoxia in the supernatant. The buildup of higher pressure differential between the primary and secondary stage filters is also problematic in as much as it promotes higher cell lysis and release of cellular factors which when active destabilize the product.

-   -   In some embodiments, a method comprises or a bioreactor is         capable of ensuring the cell-free supernatant is collected in         well aerated and mixed harvest vessel (steel or single-use). The         harvest vessel is designed to ensure it promote good surface         oxygenation through: impinging collected filtrate onto the         vessel wall surface by designing internal nozzle directed         towards the vessel wall that force the filtrate to cascade down         vessel walls; and/or ability to fill the harvest vessel before         filtrate collection with a gaseous environment composed of air         or any given blend of air and oxygen to promote oxygenation of         the collected filtrate to greater than >40% air saturation and         ≤500% air saturation.

In one embodiment, the harvest vessel is stainless steel harvest vessel. The method of supernatant collection and the design of the harvest vessel is the final element of the approach to protect the product during the harvesting step. Currently the harvested supernatant is collected in jacketed stainless steel vessels with a mixer to continually mix the collected supernatant. The harvested supernatant is discharged into the harvest vessel through headspace ports with an internal ‘J’ tube that directs the stream of supernatant onto the wall of the vessel from where it cascade down the walls to collect at the bottom of the tank. Additionally, these stainless steel vessels are filled with sterile air to keep the vessel pressurized above atmospheric pressure after they have been steam-in-place sterilized to prevent ingress of environmental contaminants across the vessel sterile boundaries. It is postulated that the harvest supernatant collected in these vessels is oxygenated as it cascades down the vessel wall under an atmosphere of pressurized air. Further oxygenation of the supernatant occurs through surface contact with the headspace above the collected supernatant.

In another embodiment, the harvest vessel is single use harvest vessel. The single use supernatant harvest vessels are gamma-irradiate and evacuated bag. During the supernatant collection the bag fills and displaces the vacuum with no gaseous headspace being formed above the collected supernatant. These bags do not have an agitator therefore collected supernatant cannot be mixed and the bags are installed into unjacketed plastic or stainless steel shells which cannot heat or cool the collected supernatant. Once the bags are filled with supernatant they are transferred into +5° C. storage for up to 14 days. The purification process may start with the primary capture step immediately after the harvest or within the 14 days hold period.

The approach to ensure the oxygenation of the production culture is maintained at a DOT in the range of greater than 40% air saturation to less than 500% air saturation during the harvesting step intends to ‘load’ the production culture with dissolved oxygen which is expected to be carried into and across the clarification step such that when the supernatant is collected it has sufficient dissolved oxygen to prevent the activation of cellular factors which may promote product dissociation. The harvest vessel is designed with to ensure the harvested supernatant continues to remain in a well oxygenated state while held under +5° C. storage for up to 14 days. This is achieved by a vessel design that ensures the supernatant continues to oxygenate while being collected and held under +5° C. storage, e.g., by surface aeration. In one embodiment, the harvest vessel design comprises the following features:

-   -   The vessel or single-used bag has an agitator/mixer that         provides sufficient axial bulk mixing to allow collected         supernatant to oxygenate through surface aeration.     -   The vessel or single use bag shell is jacketed with         appropriately sized thermo-circulator to permit the temperature         of vessel content to reach +5° C., e.g., within 4h from room         temperature and heat up to room temperature from +5° C. within         similar duration.     -   The vessel or single use bag shell is fitted with appropriately         sized air and oxygen gas mass flow controllers to permit         headspace aeration with air, oxygen or an oxygen-enriched gas.     -   The vessel or single use bag and shell are fitted with an         overlay gas filter (suitable for air and oxygen gases at flow         rate appropriate for scale of operation and an exit gas vent         filter suitable for air and oxygen gases at flow rate         appropriate for scale of operation, to permit a continuous flow         of air and oxygen across the headspace to fill the harvest         vessel before supernatant filtrate collection with a gaseous         environment composed of air or any given blend of air and oxygen         to promote oxygenation of the supernatant filtrate to greater         than >40% air saturation and ≤500% air saturation while         supernatant filtrate collection and once collection is         completed.     -   The vessel or single use bag and shell are fitted with pressure         sensor and safety interlock to stop headspace gases if pressure         exceed safety limit for the scale of operation and vessel         design.     -   The vessel or single use bag and shell are fitted with process         sensor ports for pH, DOT, Redox and temperature. The probe         location within vessel or bag allows monitoring at the lowest         operating volume appropriate for the scale of operation.     -   The vessel or single use bags are fitted with addition ports on         the top of the container with ports fitted with internal nozzles         directing flow of liquid onto the vessel wall and promoting the         liquid to cascade down the vessel wall.

Cells and Cell Culture

In one aspect, the present disclosure relates to methods and compositions for engineering or making a cell or cell line that produces a product, e.g., a recombinant product, as described herein. In another aspect, the present disclosure relates to methods and compositions for engineering or making a cell or cell line with improved, e.g., decreased dissociation, increased productivity and/or product quality.

In embodiments, the cell is a mammalian or non-mammalian cell, e.g., an insect cell, a yeast cell, a fungal cell, a plant cell, an archaeal cell, e.g., a cell from a species of Archaea, or a bacterial cell. In an embodiment, the cell is from human, mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, duck, horse, parrot, ferret, fish or cat. In an embodiment, the cell is an animal cell. In embodiments, the cell is a mammalian cell, e.g., a human cell or a rodent cell, e.g., a hamster cell, a mouse cell, or a rat cell. In an embodiment, the cell is a prokaryotic cell, e.g., a bacterial cell. In an embodiment, the cell is a species of Actinobacteria, e.g., Mycobacterium tuberculosis).

In one embodiment, the cell is a Chinese hamster ovary (CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-out cell, a CHO FUT8 GS knock-out cell, a CHOZN, or a CHO-derived cell. The CHO GS knock-out cell (e.g., GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. The CHO FUT8 knockout cell is, for example, the Potelligent® CHOK1 SV (Lonza Biologics, Inc.).

In another embodiment, the cell is a HeLa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cell), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cell, COS, e.g., COS1 and COST, QC1-3, CHO-K1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DUXB11, and CHOZN, or any cells derived therefrom. In one embodiment, the cell is a stem cell. In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

In an embodiment, the cell is any one of the cells described herein that produces a product, e.g., a product as described herein. In an embodiment, the cell is any one of the cells described herein that comprises an exogenous nucleic acid encoding a recombinant polypeptide, e.g., expresses a recombinant polypeptide, e.g., a recombinant polypeptide selected from Table 2 or 3.

In an embodiment, the cell culture (e.g., production culture) is carried out as a batch culture, fed-batch culture, draw and fill culture, or a continuous culture. In an embodiment, the cell culture is an adherent culture. In an embodiment, the cell culture is a suspension culture. In one embodiment, the cell or cell culture is placed in vivo for expression of the recombinant polypeptide, e.g., placed in a model organism or a human subject.

For example, the methods are performed in a large-scale bioreactor, e.g., a bioreactor having at least two impellers, large-scale bioreactor systems and methods for large-scale cultivation and propagation of mammalian cells. In an embodiment, the bioreactor is a bioreactor as described in 2011/0312087.

In an embodiment, the cells are cultured in a bioreactor as disclosed in U.S. Ser. No. 62/242,758, the contents of which are incorporated herein in their entirety. Disclosed therein is a system and method for control of at least one bioreactor, other cell cultivation-related equipment, and systems containing any combination of these. For example, the system and method for bioreactor control may include controlling a plurality of bioreactors and other types of cultivation-related equipment, such as equipment for fermenting, harvesting, equipment for microfiltration and purification (e.g., liquid chromatography skid system), buffer preparation, media preparation, etc. The plurality of bioreactors and the other types of cultivation-related equipment may be located in a plant, and the control of such equipment may be known as a Plant-Wide Control System (“PWCS”).

In an embodiment, the method is performed in a manufacturing facility that provides both batch and continuous manufacturing using at least one piece of single-use disposable technology, e.g., whose described in U.S. Ser. No. 62/246,478, the contents of which are incorporated herein in their entirety. In an embodiment, the manufacturing facility is for the production of active pharmaceutical ingredients (“API”).

Cooling can be effected using methods known in the art. For example, larger reactors will typically have a water jacket through which thermostatically controlled or temperature-controlled water passes to control the culture temperature. In embodiments, a process chilled water supply is delivered into the jacket to get substantial and rapid cooling. In embodiments, a refrigeration unit is used to remove heat.

Cooling can be performed at one or more desired times when culturing (i.e., causing the number of cells to increase, or induce production of a desired product from a cell culture) or cooling the production culture at the end of production phase, or when harvesting cells. Because organisms differ in the optimal temperature at which they are grown, the cooling temperature will be selected based on the relatively higher temperature of the step or steps preceding the cooling step. In embodiments, cooling of cultured cells is performed to reduce the cellular rate of oxygen consumption so that the harvest stream remains oxygenated through the harvesting process.

For mammalian cell lines, culture process temperature shifts are typically to 27-35° C. (e.g., 27, 28, 29, 30, 31 or, 32, 33, 34 or 35° C.). In some embodiments, the temperature is decreased to 30-33° C. after exponential growth phase. Without wishing to be bound by theory, it is believed that dropping the temperature to 12-18° C., e.g., 15° C., after production phase reduces the specific rate of oxygen consumption 10 fold.

In an embodiment, the culture medium is free of serum.

Other suitable media and culture methods for mammalian cell lines are well-known in the art, as described in U.S. Pat. No. 5,633,162, for instance. Examples of standard cell culture media for laboratory flask or low density cell culture and being adapted to the needs of particular cell types are for instance: Roswell Park Memorial Institute (RPMI) 1640 medium (Morre, G., The Journal of the American Medical Association, 199, p. 519 f. 1967), L-15 medium (Leibovitz, A. et al., Amer. J. of Hygiene, 78, 1p. 173 ff, 1963), Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential medium (MEM), Ham's F12 medium (Ham, R. et al., Proc. Natl. Acad. Sc. 53, p288 ff. 1965) or Iscoves' modified DMEM lacking albumin, transferrin and lecithin (Iscoves et al., J. Exp. med. 1, p. 923 ff., 1978). For instance, Ham's F10 or F12 media were specially designed for CHO cell culture. Other media specially adapted to CHO cell culture are described in EP-481 791. Other suitable cultivation methods are known to the skilled artisan and may depend upon the recombinant polypeptide product and the host cell utilized. It is within the skill of an ordinarily skilled artisan to determine or optimize conditions suitable for the expression and production of the product, e.g., the recombinant polypeptide, to be expressed by the cell.

Assays for quantifying the amount, level, or quantity of product produced or secreted, e.g., secreted into the culture media, include protein quantification assays, such as the Bradford protein assay, SDS-PAGE analysis, immunoblotting, e.g., western blot, and automated means, e.g., using a nanodrop device. Other methods for measuring increased protein production are well-known to those skilled in the art. For example, an increase in recombinant protein production might be determined at small-scale by measuring the concentration in tissue culture medium by ELISA (Smales et al. 2004 Biotechnology Bioengineering 88:474-488). It can also be determined quantitatively by the ForteBio Octet, for example for high throughput determination of recombinant monoclonal antibody (mAb) concentration in medium (Mason et al. 2012 Biotechnology Progress 28:846-855) or at a larger-scale by protein A HPLC (Stansfield et al. 2007 Biotechnology Bioengineering 97:410-424). Other methods for determining production of a product, e.g., a recombinant polypeptide described herein, can refer to specific production rate (qP) of the product, in particular the recombinant polypeptide in the cell and/or to a time integral of viable cell concentration (IVC). In an embodiment, the method for determining production includes the combination of determining qP and IVC. Recombinant polypeptide production or productivity, being defined as concentration of the polypeptide in the culture medium, is a function of these two parameters (qP and IVC), calculated according to Porter et al. (Porter et al. 2010 Biotechnology Progress 26:1446-1455). Methods for measuring protein production are also described in further detail in the Examples provided herein.

In one embodiment, the methods described herein produce a cell with improved product quality. In one embodiment, improvement of the quality of the product results in the increase, e.g., a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%, or more, increase in product quality; or a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold, or more increase, in product quality, e.g., as compared to the amount, level, or quantity of product produced by a cell not subjected to lower temperature. Such increases in product quality can be exemplified, for example, by one or more of the following:

-   -   i) an increase or decrease in disulfide bond scrambling (e.g.,         an increase or decrease the desired isoform or structure as a         result to increased or decreased disulfide bond scrambling,         e.g., for antibody molecule products).     -   ii) an increase in the amount or quantity of non-aggregated         product (or a decrease in the amount or quantity of aggregated         product);     -   iii) an increase in the amount or quantity of properly folded or         assembled product (or a decrease in the amount or quantity of         misfolded, unfolded, partially assembled, or non-assembled         product), or an increase in the ratio of properly folded or         assembled product to unfolded, misfolded, partially assembled,         or non-assembled product;     -   iv) an increase in the amount or quantity of full-length product         (or a decrease in fragmentation of the product);     -   v) an increase in the desired post-translational modifications         (or a decrease in unmodified or incorrectly modified product);     -   vi) an increase or decrease in glycan heterogeneity (e.g., for         glycosylated products);     -   vii) an increase in the amount or quantity of functional product         (or a decrease in the amount or quantity of a nonfunctional or         dysfunctional product), or an increase in the ratio of function         to nonfunctional or dysfunctional product; and/or

Methods for measuring product quality, e.g., the improvement of the product quality, of a cell or cell line generated as described herein are known in the art. In one embodiment, methods for determining the fidelity of the primary sequence of the expressed recombinant polypeptide product are known in the art, e.g., mass spectrometry. An increase in the amount or concentration of properly folded product, e.g., expressed recombinant polypeptide, can be determined by circular dichroism or assessing the intrinsic fluorescence of the expressed recombinant polypeptide. An increase in the amount or concentration of functional product can be tested using various functional assays depending on the identity of the recombinant product, e.g., recombinant polypeptide. For example, antibodies can be tested by the ELISA or other immunoaffinity assay. Other methods for determining an increase in product quality, e.g., determining aggregation, post-translational modifications, disulfide bond scrambling, can be assessed by size exclusion chromatography, high performance liquid chromatography, dynamic light scattering (DLS) approaches, and protein electrophoresis (PAGE) methods.

In some embodiments, additional steps may be performed to improve the expression of the product, e.g., transcription, translation, and/or secretion of the product, or the quality of the product, e.g., proper folding and/or fidelity of the primary sequence. Such additional steps include introducing an agent that improves product expression or product quality. In an embodiment, an agent that improves product expression or product quality can be a small molecule, a polypeptide, or a nucleic acid that encodes a polypeptide that improves protein folding, e.g., a chaperone protein. In an embodiment, the agent that assists in protein folding comprises a nucleic acid that encodes a chaperone protein, e.g., BiP, PD1, or ERO1 (Chakravarthi & Bulleid 2004; Borth et al. 2005; Davis et al. 2000). Other additional steps to improve yield and quality of the product include overexpression of transcription factors such as XBP1 and ATF6 (Tigges & Fussenegger 2006; Cain et al. 2013; Ku et al. 2008) and of lectin binding chaperone proteins such as calnexin and calreticulin (Chung et al. 2004). Overexpression of the agents that assist or improve protein folding, product quality, and product yield described herein can be achieved by introduction of exogenous nucleic acids encoding the agent. In another embodiment, the agent that improves product expression or product quality is a small molecule that can be added to the cell culture to increase expression of the product or quality of the product, e.g., DMSO. In one embodiment, maintaining the cells at a lower temperature, e.g., 1° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. lower, than the temperature that the cells are normally grown can improve the quality of the product by reducing or eliminating dissociation of the product.

Any of the methods described herein can further include additional selection steps for identifying cells that have high productivity or produce high quality products. For example, FACs selection can be utilized to select specific cells with desired characteristics, e.g., higher expression of a protein folding proteins, e.g., chaperones.

In one aspect, the disclosure provides methods that include a step for recovering or retrieving the recombinant polypeptide product. In embodiments where the recombinant polypeptide is secreted from the cell, the methods can include a step for retrieving, collecting, or separating the recombinant polypeptide from the cell, cell population, or the culture medium in which the cells were cultured. In embodiments where the recombinant polypeptide is within the cell, the purification of the recombinant polypeptide product comprises separation of the recombinant polypeptide produced by the cell from one or more of any of the following: host cell proteins, host cell nucleic acids, host cell lipids, and/or other debris from the host cell.

In embodiments, the process described herein provides a substantially pure protein product. As used herein, “substantially pure” is meant substantially free of pyrogenic materials, substantially free of nucleic acids, and/or substantially free of endogenous cellular proteins enzymes and components from the host cell, such as polymerases, ribosomal proteins, and chaperone proteins. A substantially pure protein product contains, for example, less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of contaminating endogenous protein, nucleic acid, or other macromolecule from the host cell.

Methods for recovering and purification of a product, e.g., a recombinant polypeptide, are well established in the art. For recovering the recombinant polypeptide product, a physical or chemical or physical-chemical method is used. The physical or chemical or physical-chemical method can be a filtering method, a centrifugation method, an ultracentrifugation method, an extraction method, a lyophilization method, a precipitation method, a chromatography method or a combination of two or more methods thereof. In an embodiment, the chromatography method comprises one or more of size-exclusion chromatography (or gel filtration), ion exchange chromatography, e.g., anion or cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, and/or multimodal chromatography.

In embodiments, reduction in temperature is combined with reducing temperature with reducing pH, thioredoxin inhibitors, e.g., metal ions. In embodiments, the combination treatment is performed in a bioreactor as described above.

Examples

The invention is further described in detail by reference to the following examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1. Background and Identification of Non-Essential, Redundant, and/or Secretory Endogenous Proteins Candidates for Decluttering

Chinese hamster ovary (CHO) cells are the world's primary production chassis for the generation of biopharmaceuticals (Walsh, 2014). Although huge amounts of time, effort and money are spent on developing these cells from adherent cell lines to stationary and then manipulating the cells utilising overexpression and synthetic elements to boost titre, growth rate, and cell-specific recombinant protein production rate (qP), the cellular chassis and genome effectively remains the same as that of the progenitor Chinese hamster. Because of this, the cell often behaves as if it was present within a Chinese hamster instead of an entirely artificial environment. This is exemplified by a number of seemingly functionally redundant genes still encoded and subsequently proteins expressed by the cell line. There are literature examples of genes that have successfully been knocked down or knocked out of the CHO genome, leading to enhanced cellular or product characteristics. This includes the knockdown of Cers2 and Tbc1D20 for increased specific productivity (Pieper et al., 2017), Fut8 (to remove fucosylation) Bak and Bax (for greater resistance to apoptosis) (Gray et al., 2015) as well as the removal of the endogenous CHO glutamine synthetase (Bebbington et al., 1992; Cockett, Bebbington, & Yarranton, 1990). The promise shown by the knockdown or knockout of individual genes, accompanied by recent genomic sequences for CHO (Lewis et al., 2013; Xu et al., 2011) and rapid advancements in genome editing technology (Cong, Ran, Cox, Lin, & Barretto, 2013; Ran et al., 2013) has presented the opportunity for greater screening of redundant elements within the CHO genetic code.

‘Omics-level data was utilized in the form of quantitative transcriptomics and proteomics, paired with a suite of bioinformatics tools and literature analysis, to rationally identify high abundance, non-essential proteins, predicted to compete with the target mAb for endoplasmic reticulum (ER) space as well as post translational modification (PTM) and secretory machinery. The strategy is primarily based on the analysis of Lonza RNA-seq data from a bioreactor experiment on a CHO cell line. Samples were taken and extracted at days 4, 7 and 9 and RNA-seq analysis was performed against the CHOK1GS (Ensembl accession number: CHOK1GS_HDv1) genome sequence. Utilising the transcript per million (TPM) values for each gene provided a measure of the amount each gene was expressed as a portion of the total cell transcript pool. Automatic gene ID assignment resulted in IDs for 77.5%, 77.7% and 77.6% of the total transcript pool activity genes for days 4, 7, and 9 respectively. Manual assignment added gene IDs to 18.4%, 17.8% and 18.3% of the transcript pool. This resulted in a total of 13773, 14433 and 14660 gene IDs for days 4, 7, and 9 respectively; these assignments represent IDs for 95.7%, 95.4%, and 95.8% of the total transcript pool for days 4, 7, and 9, respectively. Defined thresholds were then introduced to help categorise the gene transcripts into different abundance levels (based on Ramskold et al (2009) PLoS Comput. Biol. 2009 December; 5(12):e1000598. doi: 10.1371/journal.pcbi.1000598):

-   -   Not expressed genes (fragments per kilobase of exon model per         million reads mapped (FPKM)<0.3) (TPM <0.4)     -   Lowly expressed genes (0.3≤FPKM<3) (0.4≤TPM<4)     -   Medium expressed genes (3≤FPKM<30) (4≤TPM<40)     -   Highly expressed genes (30≤FPKM<100) (40≤TPM<133.33)     -   Very highly expressed genes (FPKM≥100) (TPM≥133.33)

Only genes that consistently met the very high abundance gene criteria across all three test days (4, 7, and 9) were taken forward (879 genes). These genes were then passed through the PANTHER classification system (http://www.pantherdb.org/) with the Mus musculus database used as the organism that analysis was carried out against. Initially only genes containing keywords that were deemed of interest, due to functional redundancy, were highlighted for further analysis (Table E1). Following this, a list of functionally vital or highly necessary keywords was generated (Table E2). Any genes that contained these keywords were subsequently removed from further analysis and in the instance of genes containing keywords from both table E1 and E2, genes were analysed in depth for consideration of evidence as to how vital they appeared to the function of a CHO cell. Following the filtering of genes through the defined criteria, 76 targets (112 when all isoforms were considered) were left.

Bioinformatic analysis was then carried out utilising 4 different tools to hone in on the genes that further fitted our criteria as secretory proteins. The programs used were:

-   -   SignalP 4.1—(default         settings—Eukaryotes)—http://www.cbs.dtu.dk/services/SignalP/     -   TargetP 1.1—(default         settings—Non-plant)—http://www.cbs.dtu.dk/services/TargetP/     -   SecretomeP 2.0—(settings changed to         Mammalian)—http://www.cbs.dtu.dk/services/SecretomeP/     -   TMHMM 2.0—(default         settings)—http://www.cbs.dtu.dk/services/TMHMM/

Further—omics level data was generated using quantitative intracellular proteomic analysis of exponential and stationary phase cells of the CHO cell line. Data detailing total protein biomass (consideration of average copy number of the protein and its molecular weight) was noted when present for target proteins.

Previously published data on quantitative proteomic analysis of extracellular proteins present within the media of CHO cell cultures (Kumar et al. 2015 J Proteome Res. 2015 Nov. 6; 14(11):4687-703. doi: 10.1021/acs.jproteome.5b00588; Park et al. 2017 Sci Rep. 2017 Mar. 10; 7:44246. doi: 10.1038/srep44246.) was utilised to try and identify common, redundant extracellular host cell (HC) proteins. Details of whether the target protein from the previously defined list was present and if so, at a high, medium or low level (criteria defined in Table E3) (NSAF—normalised spectral abundance factor, NSC—normalised spectral count) was noted.

The summarised pipeline is shown below:

-   -   1. All very high transcript throughout culture in bioreactor     -   2. All contain at least one highlighted keywords (Table E1)     -   3. All essential keywords removed unless conflicts with point 2,         where individual assessment done (Table E2)     -   4. All predicted to be secretory proteins by multiple         bioinformatic tools     -   5. Attempted matching against Lonza IC proteomics     -   6. Attempted match to previously published CHO EC abundance data     -   7. Attempted identification of glycosites and glycosites/AA         utilising Mus musculus Uniprot data     -   8. Ranked according to defined criteria (Table E4)

A further set of a-priori targets were chosen (Fibronectin—Fn1 and Heparin sulphate proteoglycans—HSPG2) due to their perceived redundancy alongside two negative targets that although matching the majority of criteria defined above, appeared highly important to a functioning CHO cell expression system (Clusterin—Clu and 14-3-3 protein epsilon—YWHAE). The final list of 20 targets for testing is shown in Table E5.

Example 2: Knockout of Endogenous Proteins and Increase in Yield of Product

Highly specific targeted knockdown of designated genes, e.g., genes encoding endogenous proteins, e.g., extracellular, secreted, and/or redundant proteins, (e.g., genes determined in Example 1, e.g., Table E5) was carried out through the use of the Cas9 nuclease, and the impact on recombinant protein production was evaluated. Utilising an enhanced specificity Cas9 (eCas9 1.1) (Slaymaker et al., 2016) 2A peptide fused to GFP permits high throughput isolation of cells with Cas9 nuclease activity potentially permitting multiplexed genome editing (Lonowski et al. 2017 Nat Protoc. 2017 March; 12(3):581-603. doi: 10.1038/nprot.2016.165).

By successfully knocking out genes encoding endogenous proteins, e.g., extracellular, secreted, and/or redundant proteins, a minimal CHO genome lacking redundant, competing proteins (e.g., proteins (i.e., mRNAs encoding proteins) competing for translation and ER/Golgi resources with product (i.e., mRNA encoding product)) can be created. This would help generate a more streamlined genome and proteome, with improved growth and production properties better suited for the demands of high titre and growth rate required for competitive generation of biopharmaceuticals.

In some embodiments, the knockout of functionally redundant secreted genes from the CHO genome will generate an enhanced cell line capable of enhanced growth rate and expression capabilities.

To initially screen the gene knockout targets identified in Table E5, each gene target was individually knocked out of an IgG4 Mab-producing clonal GS-CHOK1SV cell line, by transient transfection with a plasmid (pX458) encoding both the Cas9-GFP nuclease referred to above, as well as gene target-specific guide RNA(s) expressed from the constitutive U6 promoter. 48 hours post-transfection the heterogenous pool of transfected cells was sorted for high GFP expression (a proxy for high Cas9 nuclease activity) by FACS, with ≥0.5×10{circumflex over ( )}6 viable cells selected and pooled for further cultivation, and subsequent evaluation for growth, Mab productivity, and gene knockout success.

In detail, a GS-CHOK1V cell line making an IgG4 Mab was transiently transfected with a plasmid (pX458) containing 1) the Cas9-GFP nuclease fusion protein expressed from the CMV promoter, and 2) guide RNA(s) specific to a target gene in its genome, expressed from the constitutive U6 promoter. 48 hours post-transfection the transfectant pool was sorted for high GFP expression by FACS, with ≥0.5×10{circumflex over ( )}6 cells collected and pooled into a new flask.72 hours later the cell culture was centrifuged, the supernatant removed, and the cell pellet resuspended to a final concentration of 0.2×10{circumflex over ( )}6 viable cells/mL within a new flask, which was subsequently grown for ≥5 days before the Mab titre was determined by ValitaTITER (Valitacell, Dublin, Ireland), and the cell counts determined by automated cell counting (ViCell—Beckman Coulter, High Wycombe, UK). The percentage change in qP of FACS-sorted pools of GS-CHOK1SV cells transfected with the pX458 vector containing gRNAs specific for the indicated target genes was calculated. All data points are expressed relative to the control GS-CHOK1SV cells transfected with the pX458 expressing the Cas9-GFP nuclease fusion protein but lacking gRNAs.

Targeting of the Cas9-GFP nuclease to genes Lambl, Mfge8, Sbsn, C1S, C1R, Nid1, Dcn, B2M, and Clu, by gRNAs, increased the mean average qP of FACS-sorted pools of GS-CHOK1SV cells relative to control GS-CHOK1SV cells transfected with the pX458 vector lacking gRNAs (FIG. 1A).

To determine the success of the Cas9-GFP gene editing targeted to the specified genes, genomic DNA was extracted from the FACS-sorted cells and evaluated by TIDE analysis (Brinkman et al. 2014, Nucleic Acids Res. 2014 Dec. 16; 42(22):e168. doi: 10.1093/nar/gku936). The results demonstrated a high rate of targeted gene indels achieved by this method, ranging from ≥60 to 90% (FIG. 1B), strongly suggesting that the phenotypic readout (i.e. observed increase in qP) was a function of the gene edit in the pool.

Example 3: Multi-Gene Knockout of Endogenous Targets and Increase in Yield of a Product

The data presented in Example 2 suggests in part that several endogenous genes encoding non-essential, redundant, and/or secreted endogenous proteins in a cell can be individually knocked out to increase the yield of a Mab from a CHO cell line. As the number of endogenous proteins transiting the cellular secretory apparatus is very high, it is likely that multiple genes encoding essential, redundant, and/or secreted endogenous proteins will need to be removed in order to maximise the increase in qP obtained using this strategy. To test this, the gene targets Lambl, Mfge8, Sbsn, C1S, C1R, Nidl, Dcn, B2M, and Clu, which correlated with the highest increase in qP when knocked out individually (FIG. 1), were knocked out in random, triplicate combinations within a GS-CHOK1SV cell line using techniques of Examples 1 and 2 (Table E6), but avoiding combinations of targets that are located on the same CHO chromosome (C1S and C1R, Mfge8 and Nidl, and Dcn and Clu), so as to reduce the risk of large-scale genomic recombination. When combined in triplicate, some combinations of gene targets correlated with a substantial increase in average titre and qP, for example CIS, Dcn, and Lambl demonstrated an average increase of 25% in titre and 28% in qP when combined in a triple knockout pool, compared to control GS-CHOK1SV cells transfected with the pX458 vector lacking gRNAs (Table E7).

Example 4: Tables

TABLE E1 PANTHER assigned keywords—genes highlighted for further analysis Blood coagulation Vesicle associated Vesicle coat protein Integral to membrane Complement component Amyloid Huntington (disease) Placenta Chemokines CD Communication Bone marrow Estrogen Cell adhesion Immunity Galectin Epithelial Alzheimer Neuronal Parkinson Cytokine Embryonic Apoptosis Transmembrane receptor Secretion Exosome Endoplasmic reticulum Extracellular space Extracellular matrix Extracellular region Histocompatibility MHC Immune

TABLE E2 PANTHER assigned keywords—genes removed from further analysis Ribosomal Eukaryotic initiation ATP ADP Actin Myosin RNP NADH Ubiquitin Peptidyl Mitochondria/on Transporter Polymerase Heat shock Splicing Nucleosome RNA/DNA binding Kinase Cell division Cytochrome C Histone Transcription Translation cAMP Chaperone Folding Glycosylation Cell cycle Tubulin Kinesin Dynein Metabolic/ism Cytoskeleton Traffic Calmodulin Ras Collagen Endoplasmic Golgi

TABLE E3 Criteria for abundance level in Kumar et al (2015) and Park et al (2017) Gene ranking Criteria met 1A All in Park et al. over 1.0 in batch or fed batch highlighted. Also present in Kumar et al. 1B All in Park et al. over 1.0 in batch or fed batch highlighted. Also present in Kumar et al. 1C All in Park et al. over 1.0 in batch or fed batch highlighted. 1D All in Park et al. over 1.0 in batch or fed batch highlighted. 2A All in high abundance in Kumar et al. (NSAF < −8.89) highlighted. Also present Park et al. 2B All in high abundance in Kumar et al. (NSAF < −8.89) highlighted. Also present in Park etal. 2C All in high abundance in Kumar et al. (NSAF < −8.89) highlighted. 2D All in high abundance in Kumar etal. (NSAF < −8.89) highlighted. 3A All in Park over 0.5 in batch of fed batch highlighted. Also present in Kumar et al. 3B All in Park over 0.5 in batch of fed batch highlighted. Also present in Kumar et al. 3C All in Park over 0.5 in batch of fed batch highlighted. 3D All in Park over 0.5 in batch of fed batch highlighted. 4A All in high abundance in Kumar et al. (NSAF < −12) highlighted. Also present in Park et al. 4B All in high abundance in Kumar et al. (NSAF < −12) highlighted. Also present in Park et al. 4C All in high abundance in Kumar et al. (NSAF < −12) highlighted. 4D All in high abundance in Kumar et al. (NSAF < −12) highlighted.

TABLE E4 Final criteria considered for target ranking Protein abundance classification Protein level Kumar—High          NSAF ≥ −8.8939 Kumar—Medium −8.8939 < NSAF ≥ −12 Kumar—Low        NSAF < −12 Park—High         NSC ≥ 1.0   Park—Medium    0.5 ≥ NSC < 1.0 Park—Low         NSC < 0.5

TABLE E5 Target genome IDs Gene Added info Gene name ID Ensembl gene ID Function (see key to TABLE E5)  1. Beta-2-microglobulin B2M ENSCGRG00001018239 Component of class 1 MHC 1A, SP-Y, TP-S1, Sec-0.523, TMH-1  2. Complement C1r C1r ENSCGRG00001011539 Cellular immune system 2B, SP-Y, TP-S2, Sec-0.717, TMH-0  3. Complement C1s C1s ENSCGRG00001018041 Cellular immune system 2D, SP-Y, TP-S2, Sec-0.766, TMH-0  4. C—C motif Ccl2 ENSCGRG00001010610 Chemotactic factor that 1D, SP-Y, TP-S1, Sec-0.345, TMH-0 chemokine 2 precursor attracts monocytes  5. CD44 antigen CD44 ENSCGRG00001022042 Receptor for hyaluronic acid- 4B, SP-Y, TP-S1, Sec-0.748, TMH-0 mediates cell cell interactions  6. Clusterin Clu ENSCGRG00001021841 Extracellular chaperone that SP-Y, TP-S1, Sec-0.574, TMH-0 prevent aggregation—NEG  7. Dystroglycan Dag1 ENSCGRG00001013896 Multifunctional—basal 4A, SP-Y, TP-S1, Sec-0.068, TMH-1 membrane assembly  8. Decorin Den ENSCGRG00001019795 Binds type I and II collagen 1D, SP-Y, TP-S1, Sec-0.533, TMH-0  9. Fibronectin Fn1 ENSCGRG00001023892 Fibronectins bind cell surfaces SP-Y, TP-S2, Sec-0.363, TMH-0 and various compounds—AP 10. Follistatin-related Fst11 ENSCGRG00001019352 Modulate action of 2A, SP-Y, TP-S2, Sec-0.507, TMH-0 protein 1 precursor growth factors 11. Heparan sulphate HSPG2 ENSCGRG00001024687 Integral component of SP-Y, TP-S3, Sec-0.459, TMH-0 proteoglycan 2 basement membranes—AP 12. Intercellular ICAM1 ENSCGRG00001015045 Intercellular adhesion 4A, SP-Y, TP-S1, Sec-0.539, TMH-1 adhesion molecule 1 13. Laminin B1 Lamb1 ENSCGRG00001015692 Tissue organisation 3A, SP-Y, TP-S1, Sec-0.391, TMH-0 during embryonic dev 14. Laminin Lamc1 ENSCGRG00001012726 Migration and organisation 1A, SP-Y, TP-S3, Sec-0.238, TMH-0 subunit gamma-1 of cells into tissues 15. Lactadherin Mfge8 ENSCGRG00001011973 Aids phagocytic removal of 1C, SP-Y, TP-S1, Sec-0.769, TMH-0 apoptotic cells in tissues 16. Nidogen-1 precursor Nidi ENSCGRG00001013281 Sulfated glycoprotein in 1C, SP-Y, TP-S1, Sec-0.676, TMH-0 basement membranes 17. Prosaposin precursor PSAP ENSCGRG00001012506 Myelinotrophic and 1A, SP-Y, TP-S1, Sec-0.707, TMH-0 neurotrophic factor 18. Subrabasin precursor Sbsn ENSCGRG00001014797 Seen in epidermis, subrabasal 3C, SP-Y, TP-S1, Sec-0.733, TMH-0 keratinocytes and stomach 19. Tubulointerstitial Tinag11 ENSCGRG00001024931 Adrenocortical zonation 1A, SP-Y, TP-S2, Sec-0.893, TMH-0 nephritis antigen-like 20. 14-3-3 protein epsilon YWHAE ENSCGRG00001009921 Implicated in regulation SP-N, TP-NA, Sec-0.33, TMH-0 signalling pathways—NEG

Key for TABLE E5 Bioinformatic tool name Outcome Signal peptide (SP-) Yes (Y) OR No (N) TargetP (TP-) Localisation—Secreted (S)/unknown (NA), Reliability score 1-4 (1 = highest confidence) Secretome P (Sec-) Score over 0.6 indicates non-standard secretion in eukaryotes—only considered when no SP predicted TMHMM (TMH-) Predicted number of transmembrane helices—TMH A-priori or negative targets A-priori (AP) OR negative targets (NEG)

TABLE E6 Random Triplicate Combinations of Knockout Genes Incompatible Random Random Random Random Random Random Random Random Random Random Targets 1 2 3 4 5 6 7 8 9 10 C1s, C1r Lamb1 C1S B2m Cir C1S C1r C1S Dcn Clu B2m Mfge8, Nid1 Mfge8 Clu C1S Mfge8 Dcn Dcn Lamb1 Lamb1 Mfge8 C1r Dcn, Clu Sbsn Sbsn Lamb1 Sbsn Lamb1 Mfge8 Nid1 Nid1 Sbsn Nid1

TABLE E7 Percentage change in Mab titre, qP, and growth (integral of viable cell density, IVCD) of FACS-sorted pools transfected by pX458 containing gRNAs specific to random, triplicate combinations of target genes. All values are stated relative to control cells transfected with pX458 lacking gRNAs. MULTIPLEX GROUP % IVCD % TITER % qP Random 1 2.77 24.14 21.70 Random 2 −3.75 −12.42 −8.78 Random 3 −4.35 5.54 10.83 Random 4 −8.60 −1.81 6.55 Random 5 −1.42 24.60 27.73 Random 6 −4.17 2.16 7.26 Random 7 −2.84 2.99 4.61 Random 8 −1.45 14.23 17.19 Random 9 1.18 13.93 12.17 Random 10 −15.71 −16.00 −0.33

Example 5: References

-   Bebbington, R. C., Renner, G., Thomson, S., King, D., Abrams, D., &     Yarranton, G. T. (1992). High level expression of a recombinant     antibody from myeloma cells using a glutamine synthetase gene as an     amplifiable selectable marker. Nature Biotechnology, 10, 169-175. -   Brown, A. J., Gibson, S., Hatton, D., & James, D. C. (2018).     Transcriptome-Based Identification of the Optimal Reference CHO     Genes for Normalisation of qPCR Data. Biotechnology Journal, 13(1),     1-7. https://doi.org/10.1002/biot.201700259 -   Cockett, I. M., Bebbington, R. C., & Yarranton, T. G. (1990). High     level expression of tissue inhibitor of metalloproteinases in     chinese hamster ovary cells using glutamine synthetase gene     amplification. Nature Biotechnology, 8, 662-667.     https://doi.org/10.1038/nm0798-822 -   Cong, L., Ran, F., Cox, D., Lin, S., & Barretto, R. (2013).     Multiplex genome engineering using CRISPR/Cas systems. Science,     339(6121), 819-823.     https://doi.org/10.1126/science.1231143.Multiplex -   Gray, L. M., Lee, J. S., Gerling, S., Kallehauge, T. B., Hansen, A.     H., Kol, S., . . . Kildegaard, H. F. (2015). One-step generation of     triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent     enrichment. Biotechnology Journal, 10(9), 1446-1456.     https://doi.org/10.1002/biot.201500027 -   Kittler, R., Surendranath, V., Heninger, A. K., Slabicki, M., Theis,     M., Putz, G., . . . Buchholz, F. (2007). Genome-wide resources of     endoribonuclease-prepared short interfering RNAs for specific     loss-of-function studies. Nature Methods, 4(4), 337-344.     https://doi.org/10.1038/nmeth1025 -   Kumar, A., Baycin-Hizal, D., Wolozny, D., Pedersen, L. E., Lewis, N.     E., Heffner, K., . . . Betenbaugh, M. J. (2015). Elucidation of the     CHO super-ome (CHO-SO) by proteoinformatics. Journal of Proteome     Research, 14(11), 4687-4703.     https://doi.org/10.1021/acs.jproteome.5b00588 -   Lewis, N. E., Liu, X., Li, Y., Nagarajan, H., Yerganian, G.,     O'Brien, E., . . . Palsson, B. O. (2013). Genomic landscapes of     Chinese hamster ovary cell lines as revealed by the Cricetulus     griseus draft genome. Nature Biotechnology, 31(8), 759-765.     https://doi.org/10.1038/nbt.2624 -   Park, J. H., Jin, J. H., Lim, M. S., An, H. J., Kim, J. W., &     Lee, G. M. (2017). Proteomic analysis of host cell protein dynamics     in the culture supernatants of antibody-producing CHO cells.     Scientific Reports, 7(November 2016), 1-13.     https://doi.org/10.1038/srep44246 -   Pieper, L. A., Strotbek, M., Wenger, T., Gamer, M., Olayioye, M. A.,     & Hausser, A. (2017). Secretory pathway optimization of CHO producer     cells by co-engineering of the mitosRNA-1978 target genes CerS2 and     Tbc1D20. Metabolic Engineering, 40(October 2016), 69-79.     httpslidoi.org/10.1016/j.ymben.2017.01.003 -   Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., &     Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system.     Nature Protocols, 8(11), 2281-2308.     https://doi.org/10.1038/nprot.2013.143 -   Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., &     Zhang, F. (2016). Rationally engineered Cas9 nucleases with improved     specificity. Science, 351(6268), 84-88.     https://doi.org/10.1126/science.aad5227 -   Walsh, G. (2014). Biopharmaceutical benchmarks 2014. Nature     Biotechnology, 32(10), 992-1000. -   Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., . .     . Wang, J. (2011). The genomic sequence of the Chinese hamster ovary     (CHO)-K1 cell line. Nature Biotechnology, 29(8), 735-741.     https://doi.org/10.1038/nbt.1932

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. The invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims are introduced into another claim. Where elements are presented as lists, e.g., in Markush group format or as an alternative, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. It is also noted that the terms “comprising” and “containing” are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. 

What is claimed is:
 1. A method of manufacturing a product, e.g., recombinant polypeptide, in a cell, e.g., a production cell, the method comprising: reducing the level of an endogenous protein in the cell, e.g., production cell, thereby manufacturing the product.
 2. The method of claim 1, wherein the cell is a production cell.
 3. The method of either claim 1 or 2, further comprising culturing the cell under conditions suitable for producing the product, e.g., recombinant polypeptide.
 4. The method of any preceding claim, further comprising providing a cell, e.g., production cell.
 5. A method of manufacturing a product, e.g., recombinant polypeptide, in a cell, e.g., a production cell, the method comprising: providing a cell, e.g., production cell, reducing the level of an endogenous protein in the cell, e.g., production cell, and culturing the cell under conditions suitable for producing the product, e.g., recombinant polypeptide, thereby manufacturing the product.
 6. The method of any preceding claim, further comprising harvesting the product, e.g., separating the product from the production mixture and/or providing a purified preparation of product, e.g., by methods described herein or known in the art.
 7. A method of making a cell, e.g., a production cell, the method comprising: reducing the level of an endogenous protein in the cell, e.g., production cell, wherein the cell, e.g., production cell, is capable of expressing a product, e.g., recombinant polypeptide.
 8. The method of claim 7, further comprising providing a cell, e.g., production cell.
 9. The method of any preceding claim, wherein the endogenous protein is a non-essential protein (e.g., wherein reduction of the level of or complete loss of the protein does not significantly deleteriously effect cell viability or product production).
 10. The method of any preceding claim, wherein the endogenous protein is an extracellular protein.
 11. The method of any preceding claim, wherein the endogenous protein is a secreted protein.
 12. The method of any preceding claim, wherein the endogenous protein is localized to the Golgi apparatus.
 13. The method of any preceding claim, wherein the endogenous protein is localized to the endoplasmic reticulum (ER).
 14. The method of any preceding claim, wherein the endogenous protein is a redundant protein (e.g., wherein the functionality or activity of the protein significantly (e.g., partly or completely) overlaps with the functionality or activity of another endogenous protein present in the cell).
 15. The method of any preceding claim, wherein the endogenous protein is identified and/or selected by the method of Example 1, e.g., by association with keywords of Table E1, non-association with keywords of Table E2, and/or predicted to be secretory protein.
 16. The method of any preceding claim, wherein the endogenous protein is selected from Table E5.
 17. The method of any preceding claim, wherein the level of one, two, three, four, five, six, seven, eight, nine, ten, or more (e.g., twenty or more) endogenous proteins are decreased.
 18. The method of any preceding claim, wherein the level of endogenous protein encoded by one, two, or three genes of a combination of genes listed in Table E6 is reduced.
 19. The method of any preceding claim, wherein reducing the level of an endogenous protein in the cell comprises eliminating, e.g., knocking out, a copy of the gene encoding the endogenous protein from the genome of the cell or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 20. The method of any preceding claim, wherein reducing the level of an endogenous protein in the cell comprises eliminating, e.g., knocking out, all (e.g., both) copies of the gene encoding the endogenous protein from the genome of the cell or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 21. The method of either claim 19 or 20, wherein eliminating, e.g., knocking out, comprises introducing a deletion, substitution, or insertion mutation to the gene encoding the endogenous protein or to the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the level of expression of the endogenous protein.
 22. The method of claim 21, wherein eliminating, e.g., knocking out, comprises introducing a deletion or insertion of 1, 2, 3, 4, 5, 6, 7, 8 9, 10, or more base pairs in the gene encoding the endogenous protein or in the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 23. The method of claim 21, wherein eliminating, e.g., knocking out, comprises substituting 1, 2, 3, 4, 5, 6, 7, 8 9, 10, or more base pairs for different base pairs (e.g., transition or transversion mutations) in the gene encoding the endogenous protein or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 24. The method of any of claims 19-23, wherein eliminating, e.g., knocking out, comprises using a CRISPR-Cas9 molecule, e.g., a Cas9 molecule, in combination with a RNA (e.g., gRNA, sgRNA, and/or tracrRNA), specific for the endogenous protein encoding gene or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 25. The method of claim 24, wherein using a CRISPR-Cas9 molecule comprises: providing in the cell a CRISPR-Cas9 molecule (e.g., a Cas9 molecule), e.g., and a RNA (e.g., gRNA, sgRNA, and/or tracrRNA), specific for the endogenous protein encoding gene or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein; or providing in the cell a nucleic acid encoding the CRISPR-Cas9 molecule (e.g., a Cas9 molecule), e.g., and a RNA (e.g., gRNA, sgRNA, and/or tracrRNA), specific for the endogenous protein encoding gene or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 26. The method of any of claims 1-18, wherein reducing the level of an endogenous protein in the cell comprises using a siRNA, e.g., capable of binding to nucleic acid, e.g., mRNA, encoding the endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably linked thereto.
 27. The method of claim 26, wherein using an siRNA comprises: providing in the cell a siRNA, or providing in the cell a nucleic acid encoding the siRNA.
 28. The method of either claim 26 or 27, wherein the siRNA is designed, selected, or produced by a method selected from or equivalent to Dharmacon Horizon siDesign tool, InvivoGen siRNA Wizard, GenScript siRNA Construct Services, IDT Custom Dicer-Substrate siRNA, Sigma-Aldrich siRNA Design Service, or those taught by www.rnaiweb.com/RNAi/siRNA_Design/.
 29. A cell, e.g., production cell, capable of producing a product, e.g., recombinant polypeptide, wherein the cell, e.g., production cell, comprises a deletion, substitution, or insertion mutation to a copy of the gene encoding an endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the level of expression of the endogenous protein.
 30. The cell of claim 29, wherein the cell comprises a deletion, substitution, or insertion mutation to all (e.g., both) copies of the gene encoding an endogenous protein or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein, e.g., wherein the mutation reduces the level of expression of the endogenous protein.
 31. The cell of either claim 29 or 30, wherein the cell does not comprise a functional copy of the gene encoding the endogenous protein or the regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the endogenous protein.
 32. A cell, e.g., production cell, capable of producing a product, e.g., recombinant polypeptide, wherein the cell comprises an siRNA, e.g., capable of binding to a nucleic acid, e.g., mRNA, encoding an endogenous protein or a regulatory element, e.g., promoter or enhancer, operably linked thereto.
 33. The cell of any of claims 29-32, wherein the endogenous protein is a non-essential protein (e.g., wherein reduction of the level of or complete loss of the protein does not significantly deleteriously effect cell viability or product production).
 34. The cell of any of claims 29-32, wherein the endogenous protein is an extracellular protein.
 35. The cell of any of claims 29-32, wherein the endogenous protein is a secreted protein.
 36. The cell of any of claims 29-32, wherein the endogenous protein is a redundant protein (e.g., wherein the functionality or activity of the protein significantly (e.g., partly or completely) overlaps with the functionality or activity of another endogenous protein present in the cell).
 37. The cell of any of claims 29-32, wherein the endogenous protein is selected from Table E5.
 38. The cell of any of claims 29-37, wherein the cell further comprises a deletion, substitution, or insertion mutation to a copy of the gene encoding a second endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the second endogenous protein, e.g., wherein the mutation reduces the level of expression of the second endogenous protein.
 39. The cell of claim 38, wherein the second endogenous protein is selected from Table E5.
 40. The cell of either of claim 38 or 39, wherein the cell further comprises a deletion, substitution, or insertion mutation to a copy of the gene encoding a third endogenous protein or a regulatory nucleic acid sequence, e.g., promoter or enhancer, operably coupled to the gene encoding the third endogenous protein, e.g., wherein the mutation reduces the level of expression of the third endogenous protein.
 41. The cell of claim 40, wherein the third endogenous protein is selected from Table E5.
 42. The cell of any of claims 38-41, wherein the first and second, or first, second, and third endogenous proteins are encoded by two or three genes of a combination of genes listed in Table E6.
 43. The cell of any of claims 29-42, wherein the endogenous protein is localized in the Golgi apparatus.
 44. The cell of any of claims 29-42, wherein the endogenous protein is localized in the endoplasmic reticulum (ER).
 45. The method or cell of any preceding claim, wherein the cell, e.g., production cell, is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS, a CHO GS knock-out cell (e.g., CHO-K1SV GS knockout cell), a CHO FUT8 GS knock-out cell (e.g., Potelligent® CHOK1 SV), a CHOZN, or a CHO-derived cell.
 46. The method or cell of any of claims 1-44, wherein the cell, e.g., production cell, is a CHO-GSKO cell, a CHO Xceed™ cell CHO GS knock-out cell (e.g., GS-CHO cell), a CHO-K1SV™ GS knockout cell, or a Potelligent® CHOK1 SV.
 47. The method or cell of any of claims 1-44, wherein the cell, e.g., production cell, is a Hela, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER.C6, BHK (baby hamster kidney cell), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cell, COS, e.g., COS1 and COST, QC1-3, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, or CHOZN, or any cells derived therefrom.
 48. A product, e.g., recombinant polypeptide, produced by the method or cell of any of claims 1-47.
 49. A pharmaceutical composition comprising the product, e.g., recombinant polypeptide of claim
 48. 50. The pharmaceutical composition of claim 49, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.
 51. The method or cell of any of claims 1-47, wherein the product, e.g., recombinant polypeptide, comprises a bi-specific antibody.
 52. The method or cell of any of claims 1-47, wherein the product, e.g., recombinant polypeptide, is a protein listed in Table 1, Table 2, Table 3, or Table
 4. 53. The method or cell of any of claims 1-47, wherein the product is an antibody, e.g., a monoclonal antibody, e.g., an IgG1 antibody.
 54. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is derived from a mammalian cell, e.g., a mouse, rat, Chinese hamster ovary cell (CHO) cell, Syrian hamster, monkey, ape, dog, horse, ferret, or cat cell.
 55. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is derived from a non-mammalian cell, e.g., a cell from a duck, parrot, fish, insect, plant, fungus, yeast, bacteria. e.g., E. coli, e.g., an Archaebacteria, or an Actinobacteria.
 56. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is a stem cell.
 57. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is a differentiated form of any of the cells described herein.
 58. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is a cell derived from any primary cell in culture.
 59. The method or cell of any of claims 1-47, wherein the cell, e.g., production cell, is a CHOK1SV, GS-KO, or DUX-B11 cell.
 60. The method or cell of any preceding claim, wherein the method is suitable for use in a bioreactor, e.g., a bioreactor described herein, or the cell is capable of being cultured/grown in a bioreactor, e.g., a bioreactor described herein.
 61. A siRNA capable of binding to a nucleic acid, e.g., mRNA, encoding an endogenous protein selected from Table E5.
 62. The siRNA of claim 61, wherein the siRNA is designed, selected, or produced by a method selected from or equivalent to Dharmacon Horizon siDesign tool, InvivoGen siRNA Wizard, GenScript siRNA Construct Services, IDT Custom Dicer-Substrate siRNA, Sigma-Aldrich siRNA Design Service, or those taught by www.rnaiweb.com/RNAi/siRNA_Design/.
 63. The siRNA of either claim 61 or 62 for use in a method or cell of any of claim 1-47 or 51-60. 